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Full text of "Expression of chemokine receptors in rat central nervous system"

EXPRESSION OF CHEMOKINE RECEPTORS IN RAT CENTRAL 

NERVOUS SYSTEM 






By 

YAN JIANG 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1998 



THIS DISSERTATION IS DEDICATED TO ALL THAT I LOVE 



ACKNOWLEDGMENTS 

I would like to thank my mentor, Dr. Jeffrey K. Harrison, who was courageous 
enough to let me (someone who didn't have much experience in research) to work in his 
lab to build up my career in medical research science. During the past four years, he 
patiently guided me in every step along the way of my research from pipetting to 
scientific thinking, writing and presentation. His constant challenge prompted me to try 
harder and be better which made my years in graduate school very rewarding. I would 
also like to express my appreciation to present and former members of the laboratory for 
providing an enjoyable working environment. A special thank goes to Mina Salafranca 
for teaching me many of the techniques necessary to complete this dissertation. 

I would also like to thank every member of my committee, Drs. Wolfgang J. 
Streit, Thomas C. Rowe, John M. Petitto, and Joel Schiffenbauer. I am grateful for their 
understanding and support. Special thanks to Dr. Streit and members of his laboratory for 
generously giving me unlimited access to their knowledge, facilities and friendship. 

I want to express my gratitude to all of the faculty members, secretary staffs and 
fellow graduate students of the Department of Pharmacology and Therapeutics for 
making my years in graduate school a truly pleasant experience. I would like to show my 
appreciation to Lynn Raynor for her friendship, encouragement and enlightening 
conversations, especially during difficult days of my life. 



in 



Finally, I would like to thank my family, in particular, my husband, who has 
always been there for me throughout our ten years of marriage, and my mother, who 
taught me a principle that carries my life to a truly blessed one — a principle that says it is 
fine if you are not the best but it is detrimental if you don't try your best. 






IV 



TABLE OF CONTENTS 

page 

ACKNOWLEDGMENTS iii 

LIST OF TABLES viii 

LIST OF FIGURES ix 

ABSTRACT xi 

CHAPTERS 

1. INTRODUCTION 1 

Chemokine: Identification, Expression, and Function 1 

Identification of Chemokine Peptides 2 

Production, Expression, and Functions of Chemokines 4 

Molecular Cloning and Expression of Chemokine Receptors 7 

Molecular Cloning of CXC Chemokine Receptors 7 

Molecular Cloning of CC Chemokine Receptors 9 

Cloning of CX3CR1.XCR1 12 

Signal Transduction Properties of Chemokine Receptors 13 

Expression of Chemokines and Chemokine Receptors in the Central 

Nervous System 15 

Chemokines and Receptors Expression in EAE and Other Models for 

MS 15 

The Expression of Chemokines and Their Receptors in MS Patients 19 

Chemokines and Receptors in Other Neuropathologies 20 

Microglia — the Brain Immune Responsive Cells 23 

Chemokines, Chemokine Receptors and FflV 24 

Some Chemokine Receptors are HTV Entry Cofactors 24 

Inhibition of FHV Entry by Chemokine Ligands 26 

Chemokine Receptors and HTV in the CNS 27 

Specific Aims 28 

2. MATERIALS AND METHODS 32 

Materials 32 

Methods 33 



Polymerase Chain Reaction (PCR) Amplification of Novel DNA 

Sequences 33 

Isolation of Rat Genomic Clones 34 

Southern Blot Analysis of Rat Genomic DNA 34 

Transfection of HEK293 Cells 34 

Measurement of Intracellular Calcium Levels 35 

Expression of Rat CCR5 in Xenopus Laevis Oocytes 36 

RNA Isolation, RNase Protection Assay, and Northern Analysis 36 

Preparation of Primary Cultures of Rat Microglia 37 

Induction of Rat EAE 38 

Molecular Cloning and Sequencing of Rat Fractalkine cDNA 39 

Expression and Functional Characterization of Rat CX3CR1 in 

Transfected Cells 39 

In situ Hybridization Analysis 40 

Lectin Staining and Immunohistochemical Analysis 42 

Facial Nerve Transections 43 

Tumor Inoculation 44 

Terminal Transferase-Mediated UTP Nick End Labeling (TUNEL- 

Labeling) 44 

3. CHEMOKINE RECEPTOR EXPRESSION IN CULTURED GLIA AND RAT 

EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS 46 

Introduction 46 

Results 48 

Isolation of Rat Genomic DNA Containing Sequences Encoding Novel 

G-Protein Coupled Receptors 48 

Signal Transduction Properties of Rat CCR2 and CCR5 49 

Genomic Analysis 50 

Tissue Specific Expression of Rat CCR2 and CCR5 50 

Expression and Regulation of the Rat CCR2 and CCR5 in Cultured 

Microglia 51 

Modulation in Rat EAE 51 

Discussion 52 

4. ROLE FOR NEURONALLY-DERTVED FRACTALKINE IN MEDIATING 

INTERACTIONS BETWEEN NEURONS AND CX3CR1 -EXPRESSING 
MICROGLIA— AN IN VIVO STUDY IN THE FACIAL MOTOR NEURON 
REGENERATION PARADIGM 65 

Introduction 65 

Results 6 8 

Molecular Cloning of Rat Fractalkine 68 

Tissue Distribution of Rat Fractalkine 68 

Distribution of Rat Fractalkine in the Brain 68 

Identification of RBS1 1 as the Receptor for Rat Fractalkine 69 



VI 



Expression and Regulation of Rat CX3CR1 mRNA in Cultured 

Microglia 69 

Expression and Regulation of rat CX3CR1 In Vivo in the Facial Motor 

Nucleus After Facial Nerve Transection 70 

Expression of Rat Fractalkine in FMN After Facial Nerve Transection 71 

Discussion 71 

5. DIFFERENTIAL EXPRESSION OF CX3CR1 IN TWO RAT GLIOMA 

MODELS— ROLE FOR TGF-(3 AND CX3CR1 IN MICROGLIAL CELL 

DEATH WITHIN RAT BRAIN TUMORS 84 

Introduction 84 

Results 87 

Expression of Rat CX3CR1 in C6 Glioma 87 

Rat CX3CR1 mRNA Expression by Microglia in C6 Gliomas 87 

Different Microglial-Infiltrating Patterns Between C6 and RG2 

Gliomas 88 

Microglial Apoptosis Within C6 and RG2 Gliomas 88 

Discussion 89 

6. CONCLUSIONS AND FUTURE DIRECTIONS 97 

LIST OF REFERENCES 101 

BIOGRAPHICAL SKETCH 126 



vn 



LIST OF TABLES 

Table page 

1-1. Ligand Specificity of Chemokine Receptors 30 

3-1. Amino Acid Identities of the Rat, Murine and Human Chemokine Receptors, 
CCR2andCCR5 57 






vin 









LIST OF FIGURES 

Figure page 

1-1. Amino Acid Alignment of Representative Members of the Human Chemokine 
Superfamily 29 

1-2. Lectin (GSA I-B4) Staining of Facial Motor Nucleus Four Days After Facial 
Nerve Transection 31 

3-1. Sequence Comparison of Rat and Murine C-C Chemokine Receptors 56 

3-2. Calcium Mobilization Response to MCP-1 By HEK293 Cells Expressing Rat 
CCR2 58 

3-3. Functional Responses of Cells Expressing Rat CCR5 59 

3-4. Southern Blot Analysis of Restriction Endonuclease-Digested Rat Genomic 

DNA 60 

3-5. Rat CCR2 RNA Tissue Distribution in Rat Solid Organs and Cells 61 

3-6. Rat CCR5 mRNA Tissue Distribution in Rat Solid Organs and Cells 62 

3-7: Dose-Dependent Increase in Rat CCR5/2 mRNA by Interferon-y (IFNy) Treated 
Microglia 63 

3-8. Expression of CCR2, CCR5, CXCR4, and RBS1 1 mRNAs in Lumbar Spinal 
Cords of EAE Rats as Detected by RNase Protection Assay or Northern Blot 
Analysis 64 

4-1. Amino Acid Sequence of Rat Fractalkine (Genbank accession number R75309)..75 

4-2. Nase Protection Analysis of Fractalkine mRNA in Various Tissues of Adult Rat 76 

4-3. Ddistribution of Rat Fractalkine mRNA in Adult Rat Brain 77 

4-4. Whole Cell Radioligand Binding Analysis of CHO-CX3CR1 -Expressing Cells. .78 



IX 



4-5. Calcium Mobilization Response to Different Forms of Fractalkine by CHO Cells 
Expressing Rat CX3CR1 79 

4-6. Dose-Dependent Increases in Rat CX3CR1 mRNA by Transforming Growth 
Factor-P (TGF-p) Treated Microglia 80 

4-7. In Situ Hybridization Analysis of CX3CR1 Expression in the Rat Facial Nucleus 
After Motor Neuron Axotomy 81 

4-8. Colocalization of Rat CX3CR1 mRNA With Microglial Cells 82 

4-9: Fractalkine Expression in the Rat Facial Nucleus After Motor Neuron Axotomy 83 

5-1. Expression of Rat CX3CR1 in C6 Glioma— Northern Blot Analysis 92 

5-2. Expression of Rat CX3CR1 in C6 Glioma — In Situ Hybridization Analysis 93 

5-3. Expression of Rat CX3CR1 in Infiltrating Microglia in C6 Gliomas 94 

5-4. Lectin (GSA I-B4) Staining of RG2 (A, B) and C6 (C, D) Sections 95 

5-5. TUNEL (Terminal Transferase dUTP Nick End-Labeling) Analysis in RG2 and 
C6 Gliomas 96 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 



EXPRESSION OF CHEMOKINE RECEPTORS IN RAT CENTRAL 

NERVOUS SYSTEM 

By 

Yan Jiang 

December, 1998 
Chairman: Jeffrey K. Harrison 
Major Department: Pharmacology and Therapeutics 

Chemokines (Chemoattractive cytokines) are a fast growing family of more than 
40 structurally related proteins that are responsible for leukocyte chemoattraction and 
activation during inflammatory processes. Receptors for the chemokine peptides are 
members of the G-protein coupled receptor superfamily. 

While the expression and functional roles of chemokine and chemokine receptors 
in peripheral systems have been well characterized, related information regarding their 
role in the central nervous system (CNS) is very limited. A number of chemokine 
peptides and receptors have been found to be expressed or up regulated in 
neuropathological circumstances such as EAE (Experimental Allergic 
Encephalomyelitis), an animal model of human multiple sclerosis. 

This study examined expression characteristics of chemokine receptors in the rat 
CNS. Two genes of the rat orthologs of human CC chemokine receptors, CCR2 and 



XI 



CCR5, have been isolated. While both are expressed in a number of peripheral tissues of 
adult rat, CCR5 mRNA is also found in rat brain, spinal cord, and cultured microglia. 
IFN-y treatment of cultured microglial upregulates CCR5 expression in a dose-dependent 
manner. While CCR5 mRNA expression was elevated in EAE rats, CCR2 mRNA 
becomes detectable in the spinal cord of these diseased rats. 

We also studied the expression of RBS1 1, a chemokine receptor-like gene, which 
has recently been identified as receptor for a newly characterized chemokine termed 
fractalkine. Both fractalkine and RBS1 1 (CX3CR1) are constitutively expressed in a 
wide variety of regions of the brain. In situ hybridization analysis revealed that rat 
CX3CR1 is expressed primarily in microglia whereas its ligand, fractalkine, is expressed 
in neurons. Interactions between this ligand and receptor may provide a mechanism by 
which neurons communicate with microglia. 

Lastly, we examined the expression of fractalkine and CX3CR1 in two rat glioma 
models: C6 and RG2 gliomas. Although rat fractalkine was not detectable in either of the 
gliomas, rat CX3CR1 mRNA was detected in C6 but not RG2 gliomas. The differential 
expression of CX3CR1 in these gliomas suggests that CX3CR1 expression may be one of 
the markers of the functional state of microglia in the CNS. 



xn 



CHAPTER 1 
INTRODUCTION 

Chemokines (Chemoattractive cytokines) are a fast growing family of more than 
40 structurally related proteins that are responsible for leukocyte chemoattraction and 
activation during inflammatory processes (Luster 1998, Baggiolini et al., 1994, Baggiolini 
et al., 1997). These proteins can be divided into at least four subfamilies based primarily 
on the relative position of four highly conserved cysteine residues in their amino acid 
sequences. Receptors for some of the chemokine peptides have been identified by 
molecular cloning techniques. They are members of the G-protein coupled receptor 
superfamily. Chemokine receptors can induce a number of cellular signaling molecules 
after being stimulated by their ligands. While the expression and function of chemokines 
and their receptors have been widely explored in peripheral systems, there is very limited 
information regarding the expression and function of these molecules in the central 
nervous system (CNS). Studies carried out in this dissertation have focused on 
characterizing the expression and regulation of chemokine receptors in the rat central 
nervous system (CNS). 

Chemokine: Identification, Expression, and Function 

Chemokines are a group of proteins that are involved in leukocyte recruitment 
during inflammation. To date, this family consists of more than 40 members that can be 
divided into four subfamilies according to the arrangement of highly conserved cysteine 



residues in their amino acid sequences (Figure 1-1). By far, CC and CXC chemokine 
subfamilies are the largest among these four subfamilies. Members of both subfamilies 
have four highly conserved cysteine residues. The first cysteine forms a disulfide bond 
with cysteine 3 and the second cysteine is linked to cysteine 4 by a disulfide bridge. In 
the CXC chemokine subfamily, the first two cysteines are separated by one amino acid 
whereas in CC chemokines, the first two cysteines are adjacent to each other. Many CXC 
chemokines are chemotactic for neutrophils while most CC chemokines have chemotactic 
activity toward monocytes, macrophages, lymphocytes, eosinophils and basophils. 

Representative members of CXC chemokine subfamily are IL-8 (interleukin 8), 
PF-4 (platelet factor 4), GRO (growth-regulated oncogene) -a, -p\ -y, IP- 10 (interferon- 
inducible protein 10) and SDF-1 (stromal cell-derived factor 1). Representative members 
of the CC chemokine subfamily include the MCPs (monocyte chemoattractant protein- 1, 
-2, -3, -4, -5), the MIPs (macrophage inflammatory protein-la, 1(3, and -3), RANTES 
(regulated upon activation normal T cell expressed and secreted) and 1-309. 

Lymphotactin is the only chemokine, identified to date, that has only two 
cysteines in its amino acid sequence. It is chemotactic for lymphocytes and is designated 
as the first member of C chemokine subfamily (Kelner et al., 1994). Recently, a novel 
chemokine peptide, termed fractalkine, has been characterized that has three amino acids 
between the first two cysteine residues. It is designated as the first member of CX3C 
chemokine subfamily (Bazan et al., 1997, Pan et al., 1997). 
Identification of Chemokine Peptides 

The first identified chemokine peptide was PF-4 (Deuel et al., 1977, Hermodson 
et al., 1977, Walz et al., 1977). Because of its high affinity for heparin, this peptide was 






isolated using heparin-sepharose chromatography. Its amino acid sequence was 
determined by treatment of this peptide with different peptidases and ultimately analyzed 
by protein sequencing. PF-4 is a 70-residue peptide with a highly negatively charged N- 
terminal region, characteristic of most of the chemokine peptides. 

Ten years after the discovery of PF4, human IL-8 was identified by three 
independent groups. Walz et al. (1987) and Yoshimura et al. (1987) purified this peptide 
from conditioned media of human blood monocyte cultures treated with E.coli 
lipopolysaccharide (LPS). Simultaneously, Schmid and Weissman (1987) isolated an EL- 
8 cDNA clone from a cDNA library of SEA (staphylococcal enterotoxin A)-treated 
human peripheral blood leukocytes by the technique of differential colony hybridization 
(St. John and Davis 1979). IL-8 was found to be 35% identical to PF-4 in their amino 
acid sequences. 

Among the CC chemokine subfamily, MlP-la (Obaru et al., 1986) and MIP-1 p 
(Lipes et al., 1988) were the first to be identified. A cDNA encoding human MIP-1 a was 
isolated from a cDNA library constructed from the mRNA of TPA (12-o-tetradecanoyl 
phorbol-13-acetate) or PHA (phytoemagglutinin)-stimulated human tonsillar lymphocytes 
by differential colony hybridization. MIP-1 P was isolated in a similar manner from an 
activated T-cell library. MCP-1 is the most characterized CC chemokine. It was isolated 
by several groups using either protein purification and sequencing or sequence homology 
screening techniques (Yoshimura et al., 1989a-c, Matsushima et al., 1989, Furutani et al., 
1989, Rollins et al., 1989). In the past ten years, more than 40 CXC and CC chemokine 
peptides have been identified by techniques including protein purification and 
sequencing, sequence homology screening, or signal sequence trapping (Yoshie et al., 



1997). These peptides are generally 90-99 amino acids long with a 20-25 amino-acid 
signal sequence. 

The only member of the C chemokine subfamily, lymphotactin, was isolated from 
a complementary DNA library generated from activated mouse pro-T cells (Kelner et al., 
1 994). Its gene is located on human chromosome one. While Kelner reported its 
chemotactic activity for lymphocytes, Yoshida et al, ( 1 998) experienced difficulties in 
reproducing these results. The functional activity of this peptide has yet to be determined. 

Fractalkine, a newly identified chemokine, is the first member of the CX3C 
chemokine subfamily (Bazan et al., 1997, Pan et al., 1997, Rossi et al., 1998) and has 
some very unique properties. The full-length amino acid sequence is comprised of five 
regions: a signal peptide, a chemokine domain, a highly glycosylated mucin-like stalk 
which tethers the chemokine domain with a transmembrane domain and a short 
intracellular region. There are three amino acids in between the first two of the four 
highly conserved cysteine residues in its chemokine domain. This peptide can exist as 
both secreted soluble and membrane anchored forms. The soluble form is chemotactic 
for monocytes and lymphocytes while the membrane anchored form can promote a strong 
adhesion of these leukocytes to endothelial cells. Unlike most chemokine peptides, 
fractalkine is expressed in non-hematopoietic tissues including brain, kidney, lung, heart 
and adrenal gland. 
Production, Expression, and Functions of Chemokines 

A wide variety of cells can produce chemokine peptides including 
monocytes/macrophages, T cells, B cells, fibroblasts, endothelial cells, epithelial cells, 
keratinocytes, platelets, neutrophils, smooth muscle cells and a number of tumor cell 



lines. In the CNS, microglia, astrocytes as well as neurons have been shown to synthesize 
chemokine peptides including IL-8, MCP-1, MlP-la, MIP-ip, TCA3 (T-cell activation- 
3), and IP- 10; expression has been demonstrated from both in vitro and in vivo studies 
(Van Meir et al., 1992, Ransohoff et al., 1993, Vanguri and Farber, 1994, Huurwitz et al., 
1995, Hayashi et al., 1995, Glabinski et al., 1997, Xia et al., 1998). A number of brain 
tumor tissues can also make chemokine peptides (Desbaillets et al., 1994, Takeshima et 
al., 1994). 

Chemokines act on different types of cells such as monocytes/macrophages, T 
cells, basophils, fibroblasts, endothelial cells, keratinocytes. melanoma cells, and 
neutrophils to elicit their functions. The biological functions of chemokines include 
chemotaxis (Colditz et al., 1989, Rampart et al., 1989); changing shape of the target cells 
that is important for cell motion (Omann et al., 1987); release of histamine, elastase, p- 
glucuronidase, myeloperoxidase, and other enzymes (Kuna et al., 1992, Walz et al., 1987, 
Peveri et al., 1988); promotion of adherence (Detmers et al., 1991); induction of the 
respiratory burst (Rollins et al., 1991); and increased cell growth (Bordoni et al., 1990). 
In addition, chemokines play some roles in "normal" cellular physiology as well. They 
have been shown to be involved in such diverse processes as development, hematopoiesis 
(Nagasawa et al., 1998), angiogenesis (Strieter et al., 1995) and apoptosis (Hesselgesser 
et al., 1998). In the past few years, numerous evidence has indicated that a number of 
chemokines including MlP-la, MIP-ip, RANTES (Cocchi et al., 1995), SDF-oc (Oberlin 
et al., 1996, Bleul et al., 1996), MCP-2 (Gong et al., 1998), MDC (macrophage-derived 
chemokine) (Pal et al., 1997) and eotaxin (Choe et al., 1996) can inhibit HIV entry and/or 
viral replication within host cells. 



The expression or induction of expression of chemokines is found in a variety of 
tissues during inflammation (Luster 1998). For instance, the concentration of IL-8 and 
ENA-78 were increased in bronchoalveolar during bacterial pneumonia (Chollet-Martin 
et al., 1993, Huffhagle, 1996); MCP-1, RANTES, IP- 10 mRNA were found during 
glomerulonephritis (Tang, et al., 1995); MCP-1 and MCP-4 were seen in the plaque of 
atherosclerosis (Nelken et al., 1991, Berkhout et al., 1997); MCP-1, MP- la, eotaxin, IP- 
10, IL-8 were found to be markedly increased in inflammatory bowel disease (Garcia- 
Zepeda et al., 1996b, Grimm and Doe, 1996, Reinecker et al., 1995); RANTES, MTP-lp, 
IL-8, ENA-78 (epithelial derived neutrophil attractant) were detected in rheumatoid 
arthritis (Robinson et al., 1995, Koch et al., 1994). The appearance of different groups of 
chemokines in different inflammatory diseases where there is a subset of leukocyte 
infiltration suggests that in vivo, chemokines have the capacity to control precisely the 
type of leukocyte infiltration. 

There is a group of newly identified CC chemokines, including ELC (EBIl-ligand 
chemokine, Yoshida et al., 1997), SLC (secondary lymphoid-tissue chemokine, Nagira et 
al., 1997), TARC (thymus and activation-regulated chemokine, Imai et al., 1996), TECK 
(thymus-expressed chemokine, Vicari et al., 1997) and DC-CK1 (dendritic cell-derived 
chemokine 1, Adema et al., 1997), that are expressed constitutively in lymphoid tissues. 
All of them are found to be chemotactic for lymphocytes, including T cells, B cells or 
naive lymphocytes, but not for monocytes or neutrophils. The unique properties of these 
peptides suggest that they may constitute a new group of CC chemokines that are 
responsible for trafficking and homing of lymphocytes to particular lymphoid tissues, as 
part of their immune surveillance function. 



Molecular Cloning and Expression of Chemokine Receptors 

Many of the receptors for these chemokine peptides have been characterized by 
molecular cloning techniques (Table 1-1). They are members of the rhodopsin family of 
the G-protein coupled receptor superfamily, which are characterized by their putative 
seven hydrophobic transmembrane helices in their amino acid sequences. 
Molecular Cloning of CXC Chemokine Receptors 

To date, five human CXC chemokine receptors have been identified. Two 
receptors for EL-8 were the first chemokine receptors to be identified. In 1991, William 
Woods' group characterized a receptor by expression cloning of a human neutrophil 
cDNA library that was transfected into COS-7 cells as pools and examined for IL-8 
binding activity (Holmes et al., 1991). The pure clone obtained encodes a 350-amino- 
acid protein that not only binds to, but also responds to IL-8, to form a transient 
intracellular calcium increase in transfected cells. Its amino acid sequence was found to 
be most closely related to two other G-protein coupled receptors, the fMLP and C5a 
receptors. On the other hand, Philip Murphy's group (Murphy and Tiffany, 1991 ) 
identified another IL-8 receptor gene in an attempt to isolate the human ortholog of a 
rabbit N-formyl peptide receptor (F3R). They designed an oligonucleotide probe 
corresponding to nucleotides of the cDNA sequence of F3R to screen the cDNA library of 
a leukemia cell line (HL-60). The isolated gene contained a 1065-bp open reading frame 
(orf), encoding a 355-amino-acid protein. It is 77% identical to the other IL-8 receptor. It 
can bind to IL-8 and several other CXC chemokines including MGSA (melanoma growth 
stimulatory activity)/GRO and NAP (neutrophil activation protein)-2. Application of 



8 



these peptides to Xenopus oocytes that express this receptor triggered an intracellular 
Ca 2+ mobilization (Murphy et al., 1991). These two IL-8 receptors were later designated 
as CXCR1 and CXCR2. Both are expressed on neutrophils. CXCR2 is also found in 
myelomonocitic cell lines and its expression can be stimulated by G-CSF (granulocyte 
colony stimulating factor) and TNF (tumor necrosis factor)-a (Sprenger et al., 1994). 

RT-PCR technique was used to isolate human CXCR3. Degenerate 
oligonucleotide primers were designed based on the sequence of transmembrane domain- 
2 (TM2) and TM7 of other CXC and CC chemokine receptors (Loetscher et al., 1996). 
This receptor gene encodes a protein of 368 amino acids with putative seven 
transmembrane domains. The CXC chemokines Mig (monokine induced by interferon-y) 
and IP- 10 were identified as ligands for this receptor because of their ability to stimulate 
Ca" + mobilization and chemotaxis of receptor transfected cells. Northern blot analysis 
revealed that this receptor was expressed in T cells stimulated with EL-2. Recently, 
another CXC chemokine I-TAC (Interferon-inducible T cell Alpha Chemoattractant, 
Cole et al., 1998) was found to be the ligand of human CXCR3 and a CC chemokine 
peptide 6Ckine (Soto et al., 1998) was shown to be the ligand for murine CXCR3. 

In 1994, Loetscher et al. identified an "orphan" seven transmembrane domain 
receptor that was expressed in neutrophils, activated T cells and some leukemia cell lines. 
This receptor was later found to be a co-receptor for T-cell line tropic HTV entry (Feng et 
al., 1996). Two years later, a CXC lymphocyte chemoattractant, SDF-1 (stromal cell- 
derived factor 1), was proved to be the ligand of this receptor (Bleul et al., 1996 and 
Oberlin et al., 1996). According to the accepted nomenclature of chemokine system, it 
was designated as human CXCR4. Two CXCR4 "knock out" studies reported most 



recently (Zou et al., 1998, Tachibana et al., 1998) suggest that CXCR4 plays a very 
important role in embryonic development of the cerebellum as well as vascularization of 
the gastrointestinal tract. 

Human CXCR5 was also isolated as an orphan receptor that was expressed in B 
cells and Burkitt's lymphoma tissue (Dobner et al., 1992). Forster et al. (1996) provided 
evidence indicating the involvement of this receptor in B cell migration and localization 
by using CXCR5 "knock out" mice. Recently, Gunn et al. (1998) and Legler et al. (1998) 
characterized a CXC chemokine BLC (B-lymphocyte chemoattractant)/BCA-l (B cell- 
attracting chemokine 1) that can bind and stimulate Ca" + mobilization and chemotaxis of 
cells transfected with the CXCR5 gene. 
Molecular Cloning of CC Chemokine Receptors 

To date, ten human CC chemokine receptors have been characterized by different 
groups. Neote et al. (1993) and Gao et al. (1993) discovered human CCR1 gene using 
sequence homology cloning techniques in their attempt to isolate chemokine receptors 
that are closely related to IL-8 receptors and the fMLP receptor. Specific binding and 
Ca 2 * mobilization analysis revealed that RANTES, MIP-lcc are ligands of this receptor. 
CCR1 mRNA was detected in B-lymphocytes and some human myeloid precursor cell 
lines e.g. HL-60, U937 and THP-1. Additional chemokine peptides have been 
characterized in recent years; MCP-2 (Gong et al., 1997), MCP-3 (Combadiere et al., 
1995b), Leukotactin-1 (Youn et al., 1997), and Hemofiltrate C-C chemokine (HCC)-l 
(Tsou et al., 1998) have since been identified as ligands of CCR1 . 

Human CCR2a and CCR2b are MCP-1 receptors with alternatively spliced 
carboxyl tails (Charo et al., 1994). RT-PCR technique was performed using mRNA from 



10 

a monocytic cell line as the template to isolate these receptor genes. Degenerate primers 
were designed based on conserved sequences of CCR1, CXCR1 and CXCR2. Northern 
blot analysis indicated that both CCR2 receptors are expressed in monocytes and THP-1 
and MonoMac 6 cell lines. While MCP-1 is the only known ligand for CCR2a, MCP-2, 
MCP-3 and MCP-4 are found to be additional ligands for CCR2b (Gong et al., 1997, 
Combadiere et al., 1995b, Godiska et al., 1997). 

Similarly, CCR3 was cloned by several different groups (Combadiere et al., 
1995a, Ponath et al., 1996, Daugherty et al., 1996, Kitaura et al., 1996). This receptor is 
expressed in eosinophils and basophils (Uguccioni et al., 1997) and weakly in monocytes 
and neutrophils. It is the only receptor, to date, for eotaxin-1 and eotaxin-2 (White et al., 
1997). However, RANTES, MCP-1 (Daugherty et al., 1996), MCP-2 (Heath et al., 
1997), MCP-3 (Ponath et al., 1996), MCP-4 (Garcia-Zepeda et al., 1996a) Leukotactin- 1 
(Youn et al., 1997) are also ligands for CCR3. 

Human CCR4 was isolated from a basophil cell line, whose amino acid sequence 
shares only 49% identity to CCR1 (Power et al., 1995). It is expressed at high levels in 
thymus, T cells, B cells, monocytes, platelets, and to a lower level in spleen. The 
identification of its ligand(s) has been controversial. While Power et al. demonstrated 
detectable Ca 2+ dependent CI" currents in CCR4 expressing oocytes by MlP-la, MCP-1 
and RANTES application, Imai et al. (1997a) failed to stimulate Ca 2 ~ mobilization in 
CCR4 expressing 293/EBNA cells with these ligands. However, they successfully 
identified TARC (thymus and activation-regulated chemokine) as a specific ligand for 
CCR4. Recently, Imai et al. (1998) demonstrated that MDC (macrophage-derived 
chemokine) is also a ligand for CCR4. 



11 

A number of different groups (Samson et al., 1996a, Raport et al., 1996, 
Combadiere et al., 1996) isolated CCR5 by RT-PCR technique. These investigators were 
able to identify MP- la, MIP-lp, and RANTES as its ligands. Gong et al. (1998) 
provided evidence demonstrating that MCP-2 is also a ligand for CCR5. This receptor 
was found to play an important role in macrophage-tropic HTV-1 infection as a fusion 
cofactor (Deng et al., 1996, Dragic et al., 1996, Alkhatib et al., 1996). 

Human CCR6 (Zaballos et al., 1996, Liao, et al., 1997a), CCR7 (Schweickart et 
al., 1994), and CCR8 (Zaballos et al., 1996, Napolitano et al., 1996, Samson et al., 
1996b) were isolated via RT-PCR and initially reported as orphan receptors. LARC 
(liver and activation-regulated chemokine)/MIP-3a/Exodus-l was found to be the ligand 
of CCR6 (Baba et al., 1997, Power et al., 1997, Greaves et al., 1997, Liao et al., 1997b). 
This receptor is expressed in spleen, lymph node, gut, pancreas and peripheral blood 
lymphocytes, monocytes/macrophages. Two CC chemokine peptides, MIP-3p7CKb- 
1 1/ELC (EBI1 -ligand chemokine) and SLC (secondary lymphoid-tissue chemokine), can 
specifically bind to CCR7 expressing cells and stimulate a transient increase of the 
intracellular Ca 2+ level (Yoshida et al., 1997, Willimann et al., 1998). A number of 
lymphoid organs such as tonsil, spleen, and lymph node constitutively express CCR7. 
This receptor was also found in several B- and T-cell lines but not in the myeloid cell 
lines. Northern blot analysis was carried out on different tissue samples to determine the 
expression of CCR8. CCR8 mRNA was detectable in spleen, lymph nodes and very 
weakly in appendix and lymphocytes. To date, 1-309 is the only ligand found to be able 
to interact with CCR8 expressing cells and stimulate Ca 2+ mobilization (Roos et al., 1 997, 
Goyaetal., 1998). 



12 

Choe et al. (1998) have presented data demonstrating that human CCR9 can serve 
as a coreceptor for HTV- 1 and SIV entry. However, they did not present in detail how this 
receptor was cloned and its amino acid sequence, neither did they discuss the tissue 
distribution pattern of this receptor. Human CCR10 was identified by screening a human 
lung cDNA library with a "P labeled probe containing known EST (Expressed Sequence 
Tag) #149079 sequence (Bonini et al., 1997). The resulting cDNA contains a coding 
region of l,152bp for a 384-amino-acid protein. Its amino acid sequence is around 30% 
identical to the other CC chemokine receptors. MCP-1, MCP-3, MCP-4 and RANTES 
can bind to CCR10 expressing COS-7 cells in nanomolar concentration level. However, 
binding of MCP-3 to the receptor did not stimulate a transient increase of the intracellular 
Ca + levels. Thus, the real ligand(s) for CCR10 remains to be determined. 
Cloning of CX3CRLXCR1 

The receptor for fractalkine, designated as CX3CR1, was identified recently (Imai 
et al., 1997b). Previously, it was considered to be an orphan receptor (Harrison et al., 
1994, Raport et al., 1995, Combadiere et al., 1995c). It was found in many lymphoid cell 
lines and is broadly expressed in a variety of tissues including many discrete regions of 
the brain. 

Most recently, the receptor for lymphotactin, designated as XCR1 , has been 
identified by Yoshida et al. (1998). It is expressed in placenta and weakly in spleen and 
thymus among various human tissues. Lymphotactin binds to XCR1 and triggers an 
intracellular Ca" + increase and stimulates chemotaxis of XCR1 expressing cells. The 
functional target of lymphotactin remains to be determined because of the controversial 
results between two groups (Kelner et al., 1994, Yoshida et al., 1998). 



13 

Finally, there is an interesting chemokine receptor termed Duffy antigen receptor 
for chemokines (DARC) which interacts with most of the CC and CXC chemokines but 
does not signal (Chaudhuri et al., 1993, Horuk et al., 1993). This gene encodes a 338- 
amino-acid protein that also contains seven hydrophobic transmembrane regions 
conserved in all G-protein coupled receptors. Its function is likely to serve as reservoir of 
chemokines in the body. 

The murine and rat orthologs of many of these human chemokine receptors have 
also been identified (Bozic et al., 1994, Cerretti et al., 1993, Harada et al., 1994, Heinrich 
and Brano, 1995, Lee et al., 1995, Dunstan et al., 1996, Nagasawa et al., 1996, Heesen et 
al., 1996, Gao and Murphy, 1996, Hoogeworf et al., 1996, Post et al., 1995, Boring et al., 
1996, Kurihara and Brano, 1996, Gao and Murphy, 1995, Meyer et al., 1996, Schweickart 
et al., 1994, Zingoni et al., 1998), (Table 1). These murine and rat orthologs will be useful 
in studying the expression, regulation and functional role of chemokines and chemokine 
receptors in vivo under physiological and pathological conditions using a number of 
murine or rat animal models of human diseases. 

Signal Transduction Properties of Chemokine Receptors 

Chemokines act on their receptors to stimulate a number of signal transduction 
pathways to elicit their functions. Almost all receptors stimulate a transient intracellular 
calcium increase upon activation. In most cases, except for MCP-2, this response can be 
abolished by pertussis toxin pretreatment (Baggiolini et al., 1997, Sozzani et al., 1994) 
suggesting a Gi protein involvement in their signal transduction pathways. 
Complementation experiments in cotransfected COS-7 cells indicated that CXCR1 and 



14 

CXCR2 can couple to many G-protein subtypes including Gi2, Gi3, G14, G15, and G16 
(Wu et al., 1993). Kuang et al. also examined the selective G protein coupling by CC 
chemokine receptors in cotransfected COS-7 cells (1995). Their data revealed that while 
both CCR1 and CCR2a couple to G14, only CCR2a couples to G16. Neither CCR1, 
CCR2a nor CCR2b couple to Gq/1 1. A similar study using both COS-7 and HEK-293 
cell lines suggests that receptor-G-protein interactions are highly cell type specific (Arai 
and Charo, 1996). The G^y subunits of Gi proteins have been shown to activate specific 
isoforms of PLCP (Wu et al., 1993, Kuang et al. 1995) and mediate chemotaxis (Neptune 
and Bourne, 1997 and Arai et al., 1997). When human neutrophils are activated by IL-8, 
but not GRO-oc or NAP-2, there is a stimulation in phospholipase D activity in these cells 
which is accompanied by the increase of respiratory burst (L'Heureux et al., 1995, Jones 
et al., 1996). RANTES has also been shown to be able to stimulate phospholipase D 
activity in Jurkat cells (Bacon et al., 1998). The inhibition of cAMP (Shyamala et al., 
1997), induction of arachidonic release (Locati et al., 1994) and stimulation of inositol 
phosphate formation (Arai et al., 1996) have been well documented in chemokine 
receptor transfected cell systems when exposed to the chemokine peptides. Bacon et al. 
have demonstrated that the induction of mobilization of Ca 2+ in T cells by RANTES is 
mediated by both a G protein coupled pathway and activation of protein tyrosine kinases 
(1995). Other studies have also provided evidence of the activation of a number of 
different tyrosine kinases when the cells are treated with chemokine peptides (Schraw and 
Richmond, 1995, Bacon et al., 1996, Ganju et al., 1998). Studies from two groups 
revealed that chemokines can stimulate the activation of MAP-kinases (Jones et al., 1995, 
Knall et al., 1996), phosphoinositide 3 '-kinases (Knall et al., 1997, Turner et al., 1995), 



15 

and protein kinase B (Tilton et al., 1997) in the chemokine receptor expressing cells. 
Recently, Wong et al. reported the activation of STATs (Signal Transducers and 
Activators of Transcription) in T cells by RANTES and MP- let (Wong et al., 1998). 

In conclusion, different chemokine peptides use different G proteins in different 
cells to stimulate different signal transduction pathways for different cellular responses. 
However, more studies are needed in order to understand, more completely, the signal 
transduction pathways mediated by chemokines and chemokine receptors. 

Expression of Chemokines and Chemokine Receptors in the Central Nervous System 

While the expression and functional roles of chemokines and their receptors have 
been widely explored in the periphery, there is very limited information available 
regarding the expression and function(s) of chemokines and chemokine receptors in the 
CNS. Most of this information has been collected from studies on experimental allergic 
encephalomyelitis (EAE), the best known laboratory animal model of human multiple 
sclerosis (MS). 
Chemokines and Receptors Expression in EAE and Other Models for MS 

EAE is an inflammatory demyelinating disease of the central nervous system, 
which has many clinical and pathological features in common with multiple sclerosis 
(MS). In 1933, Rivers and his coworkers discovered that immunization of susceptible 
animals with brain tissue could induce an acute or chronic encephalomyelitis. This model 
has evolved considerably over the last few decades. EAE can be produced in monkeys, 
mice, rats, guinea pigs and rabbits using a variety of antigen preparations, including 
whole spinal cord, MBP (myelin basic protein) (Kies et al., 1960), PLP (proteolipid 



16 

protein) (Madrid et al., 1982), and peptides of these proteins (Pettinelli et al., 1982, 
Touhy, 1994). EAE can also be transferred via intravenous injection of lymphocytes 
derived from sensitized donors (Paterson, 1960) or MBP-reactive cells (Ben Nun et al., 
1981). 

Pathological findings of EAE animals include intense inflammation and some 
demyelination of brain and spinal cord. Clinical signs occur within 9-15 days after 
injection, which consist of weight loss, lethargy, loss of tail tonicity, different degrees of 
paraparesis, and incontinence. 

Because of the extensive inflammation involved in EAE, it has become a very 
useful animal model for studying the expression and function of cytokines, including 
chemokines, in the CNS. In 1993, Ransohoff et al. demonstrated expression of the 
chemokine peptides MCP-1 and IP- 10 in the spinal cords of EAE mice using RT/PCR 
and in situ hybridization analysis. Their data suggested that astrocytes were the source of 
MCP-1 and IP- 10 mRNA. At the same time, Berman's group demonstrated expression of 
MCP-1 mRNA and protein in spinal cords of EAE rats. MCP-1 mRNA levels were 
elevated immediately before the onset of clinical signs, peaked with the height of clinical 
disease, and declined with resolution of the disease (Hulkower et al.. 1993). Later, they 
defined the localization of MCP-1 by immunohistochemistry and in situ hybridization 
(Berman et al., 1996). Their data indicated that at the onset of inflammation, prior to 
clinical signs, MCP-1 was detectable in lymphocytes and endothelial cells in 
subarachnoid locations whereas after the onset of the clinical signs, MCP-1 was widely 
distributed in the spinal cord in lymphocytes, macrophages, astrocytes and endothelial 
cells. 



17 ' 

Godiska and colleagues examined the expression of a number of chemokines in 
EAE mice using RT/PCR and Northern blot analysis and found that mRNAs encoding 
RANTES, MP- la, MP- IB, TCA3, IP- 10, MCP-1 KC/GRO and MCP-3 were expressed 
in the spinal cords of EAE mice. Also, SDF was expressed in normal as well as diseased 
spinal cords and no expression of MIP-2 was detected in either normal or diseased tissue 
(1995). Using in situ hybridization analysis combined with immunohistochemistry, 
Miyagishi et al. identified the cellular localization of RANTES and MTP-1B in rat EAE. 
Their evidence indicated that mRNAs for RANTES and MP- 113 were mostly expressed 
by CD3-positive T-cells located around blood vessels. These mRNAs were also co- 
localized with GFAP-positive astrocytes as well as OX42-positive 
macrophages/microglia (1997). They were able to detect MIP-lcc expression in the spinal 
cord by day 10 post injection, but they failed to identify its cellular colocalization due to 
the fact that the combination of immunohistochemistry and in situ hybridization was 
much less sensitive using MP- la probes. 

Glabinski and colleagues examined the time course of the expression of five 
chemokines (MCP-1, MP- la, KC/GRO-a, IP- 10, and RANTES), during spontaneous 
relapses of chronic EAE mice (1997), using RT-PCR, ELISA, and in situ hybridization 
analysis. They found that all five chemokines were simultaneously up regulated during 
relapses in the brain and spinal cord, in addition, expression was confined to the CNS. In 
situ hybridization revealed that IP- 10 and MCP-1 are expressed by GFAP-positive 
astrocytes. The nuclear morphology and location of KC-expressing cells suggested 
astrocytes as the cellular expressions of this chemokine. MlP-la and RANTES are 
produced exclusively by mononuclear leukocytes in perivascular cuffs. Interestingly, 






18 

while other groups failed to demonstrate any expression of MIP-2. a CXC chemokine 
peptide, Adamus et al. (1997) were able to detect MIP-2 mRNA expression in Lewis rats 
at day 10 after MBP injection, the time of disease onset. Their in situ hybridization data 
suggested that MCP-1 was expressed in astrocytes and infiltrating cells in the white 
matter. 

The involvement of chemokines in EAE development became evident when 
Karpus et al. (1995) demonstrated that injection (i.p.) of an antibody towards MlP-la to 
SJL/J mice passively transferred with EAE can prevent the development of clinical 
disease as well as infiltration of mononuclear cells into the CNS. Antibody therapy could 
also be used to ameliorate the severity of ongoing clinical disease of these EAE mice. 
However, in their report, they didn't see the production of MCP-1 and MIP-2 peptides by 
ELISA in diseased animal spinal cord, anti-MCP-1 failed to prevent the disease 
development. In another study with the attempt to intervene the development of EAE, 
Wojcik et al. (1996) revealed that intrathecal administration of antisense oligonucleotides 
to MBP-injected Lewis rats could significantly reduce EAE scores at the height of clinical 
disease. 

Recently, Lane et al. (1998) examined the expression of a number of chemokines 
using another model, virus-induced encephalomyelitis. Transcripts of CRG-2 (cytokine- 
response gene-2)/IP-10, RANTES, MCP-1, MCP-3, MIP-lp\ and MIP-2 were seen in the 
brain of mouse hepatitis virus-infected mice 3 days postinfection. These transcripts were 
increased markedly in brains and spinal cords at day 7, which coincides with the 
occurrence of acute viral encephalomyelitis. Their in situ hybridization data confirmed 
that CGR-2/EP-10 was expressed by astrocytes. 



19 

The Expression of Chemokines and Their Receptors in MS Patients 

A number of studies have focused on examining the expression of chemokines in 
CNS tissue and cerebrospinal fluids (CSF) of MS patients or cell lines derived from MS 
patients. Miyagishi et al. (1995) observed an increase of MTP-la concentration in CSF of 
MS patients by ELISA. Based on this evidence, Bennetts et al. (1997) looked at the 
potential protection effect of CCR5 (one of the receptors for MlP-la) deletion mutation 
against MS. Two CCR5 deletion mutation individuals have been diagnosed with MS. 
This suggested that the absence of CCR5 is not protective against MS. It is likely that 
other MTP-la receptors are responsible for MTP-la related pathogenesis in MS patients. 

Since the demyelination process that occurs in MS patient is, in part, due to an 
inflammatory response in which CD4+ and CD8+ T cells and macrophage infiltrate white 
matter, Biddison et al. (1997, 1998) did a series of in vitro studies demonstrating that 
CD8-*- T cell lines derived from MS patients are capable of secreting MTP-la, MTP-1(3, 
and IP- 10. Soluble products of these CD8+ T cells are chemotactic for CD4+ T cells. 
Antisera treatment revealed that the majority of this chemotactic activity is mediated by 
IP- 10. Another study using post-mortem tissue samples of MS patients revealed that the 
expression of RANTES mRNA level in MS brain is higher than control brain. In situ 
hybridization analysis indicated that RANTES mRNA localization was associated with 
perivascular T cells (Hvas et al., 1997). These data further suggested that chemokines 
play a proinflammatory role in the pathogenesis of MS. 



20 

Chemokines and Receptors in Other Neuropathologies 

The expression of chemokines was also characterized in a number of other 
neuropathological conditions. A traumatic injury to adult mammalian central nervous 
system results in reactive astrogliosis and the migration of hematogenous cells into the 
damaged neural tissue. Several groups looked at expression of chemokines in models of 
mechanical injury of the brain based on the hypothesis that chemokines might play an 
important role in this cellular accumulation after the injury. Glabinski et al. (1996) 
identified the expression of MCP-1 mRNA in mouse brain after penetrating mechanical 
injury within 3 hours postinjury of the brain. MCP-1 mRNA levels stayed high 12 hours 
after injury and came back to base line around 24 hours postinjury. Using ELISA, they 
revealed that MCP-1 protein level was elevated in the homogenates of the lesioned tissue 
by 12 hours postinjury and approached baseline by 48 hours. Their in situ hybridization 
analysis again showed that MCP-1 was expressed by GFAP (glial fibrillary acidic 
protein) expressing astrocytes. In another study, Berman et al. (1996) also found the 
upregulation of MCP-1 mRNA and protein level in the rats subjected to stab wounds of 
the cortex. However their immunohistochemistry data indicated that MCP-1 was 
expressed by macrophages and endothelial cells from morphological assessment. 
Ghirnikar et al. (1996) found the expression of two other chemokines; namely RANTES 
and MIP-1 p\ in the rat stab wound brain by immunohistochemical staining. Double- 
labeling studies showed that MIP-1 p, but not RANTES, was expressed by astrocytes and 
macrophages near the lesion site. 

Hausmann et al. (1998) compared the type of chemokine expression between 
cortical lesions under sterile conditions or with LPS (lipopolysaccharide) injection. Only 



21 

MCP-1 expression was detectable in the sterile lesion. However, in the lesioned tissues 
of LPS injection, they found expression of mRNAs of all chemokines examined, 
including MCP-1, MEM a, NflP-ip, RANTES, IP- 10 and KC. They also examined the 
leukocytes and macrophage densities after the lesion and revealed that 
macrophage/microglia accumulation occurred more rapidly; activated microglia further 
from the lesion border; and more cells accumulated in LPS injected injury than sterile 
injury. This indicated that chemokines are instrumental in the initiation of repair 
processes following brain injury. Infusion of MCP-1 antisense oligonucleotides to the 
stab wound injured rats not only reduced MCP-1 protein expression but also decreased 
macrophage accumulation in the lesioned area (Ghirnikar, et al., 1998). This data more 
directly suggested a functional role of MCP- 1 in macrophage chemoattraction after 
mechanical brain injury. 

Focal cerebral ischemia of rat induced by middle cerebral artery occlusion 
(MCAO) is a frequently used animal model for ischemic stroke. Ischemic brain injury 
secondary to an arterial occlusion is characterized by acute local inflammation. Using 
this model, Liu et al. (1993) detected CINC/GRO/KC mRNA expression in the ischemic 
tissue. Gourmala et al. (1997) identified MCP-1 mRNA expression in the ischemic tissue 
of the rat after MCAO. Recently, Kostulas et al. (1998) examined expression of IL-8 in 
the peripheral blood of healthy individuals or patients with ischemic stroke. They found 
that plasma IL-8 concentration was increased in patients that suffered a stroke. This 
correlated positively to IL-8 mRNA expression in mononuclear cells of examined 
patients. 



22 

Another useful model for stroke is reperfusion after a transient brain ischemia 
because reperfusion after ischemia is a frequently encountered clinical condition that 
often causes greater tissue damage than persistent ischemia itself. Using a rabbit 
reperfusion model, Matsumoto et al. ( 1 997) revealed that anti-EL-8 antibody significantly 
reduced brain edema and infarct size caused by reperfusion after ischemia in comparison 
to rabbits receiving a control antibody. 

In patients suffering from pyogenic meningitis, there is a significant elevation of 
levels for IL-8, GRO-ct, MCP-1, and MIP-lp in the CSF in comparison with 
noninflammatory CSF of control individuals (Spanaus et al., 1997). The CSF of these 
patients was chemotactic for neutrophils and monocytes. A number of chemokine genes 
including CRG-2/IP-10, RANTES, MCP-1, MIP-lp, MCP-3, lymphotactin, CIO, Mff-2 
and MIP- 1 a were found to be induced in the brains of mice with lymphocytic 
choriomeningitis (Asensio and Campbell, 1997). In a study using an osmotic stimulation 
model, Koike et al. (1997) found CINC gene expression in the rat hypothalamus. In an 
excitotoxic injury model, MCP-1 was found to be expressed in the brain of intra- 
hippocampal NMDA injected postnatal rats (Szaflarski et al., 1998). 

Expression of chemokine receptors in the CNS have been reported. Horuk et al. 
(1997) examined the protein expression of DARC, CXCR1 and CXCR2 in various 
regions of the brain by immunohistochemical staining. Their data revealed that DARC 
was expressed exclusively by Pukinje cells in the cerebellum. While CXCR1 was not 
expressed in the CNS, CXCR2 was expressed at high levels by subsets of projection 
neurons in diverse regions of the brain and spinal cord. CXCR2 was also found in the 
neuritic portion of plaques surrounding deposits of amyloid of patients with Alzheimer's 



23 

disease (AD, Horuk et al., 1997). Immunohistochemical analysis has revealed that CCR3 
and CCR5 are expressed on microglia in normal and AD brains (Xia et al., 1998). 
However, not all ligands of these two receptors are found in the brain. MIP-ip was found 
predominantly in a subpopulation of active astrocytes, which were more widespread in 
AD than control brains. MlP-la appeared to be constitutively expressed at low levels by 
neurons and microglia in both AD and control brain. Eotaxin, RANTES and MCP-3 
immunoreactivity were not detected. Recently, Spleiss et al. (1998) found induction of 
CCR5 mRNA in the lesioned region of rats that underwent brain ischemia or LPS 
injection. 
Microglia — the Brain Immune Responsive Cells 

Microglia are the immune responsive cells in the central nervous system. Thus, it 
is conceivable that function of chemokines and receptors in the CNS could relate to their 
expression in microglia. There is evidence demonstrating the expression of some 
chemokines and chemokine receptors in microglia. These molecules include IL-8 
(Lipovski et al., 1998), IP- 10, MCP-1 (Sun et al., 1997), MlP-la, MP 1-p, RANTES 
(Janabi et al., 1998), SDF-p (Ohtani et al., 1998), CCR3, CCR5, and CXCR4 (Shieh et 
al., 1998). To study microglia activation in vivo, the facial nerve regeneration model is a 
very useful animal model and has been well characterized. A transection of facial nerve 
in the periphery stimulates a response in the facial motor nucleus. This response involves 
microglia proliferation and activation. Facial motoneuron cell bodies are ensheathed by 
these activating microglia (Figure 1-2). The advantage of this model is that one can 
generate a microglia response without breaking the blood brain barrier so that only the 



24 

endogenous cells are responsible for the change. This model is very useful in our studies 
examining the expression of chemokines and chemokine receptors in the CNS. 

In conclusion, by far, most studies in regard to chemokines and chemokine 
receptors in the CNS still remain at a descriptive level. However, these data implicate 
important role(s) of chemokines and their receptors in a broad variety of 
neuropathological conditions. 

Chemokines, Chemokine Receptors and HTV 

In the past two years, chemokines and chemokine receptors have been brought to 
the spotlight largely because of the discovery of the functional role chemokines and 
chemokine receptors play in HTV (Human immunodeficiency virus) infection. 
Some Chemokine Receptors Are HTV Entry Cofactors 

The primary interaction site between HTV- 1 and infectable cells is the CD4 
molecule. This interaction is mediated by the viral surface glycoprotein gpl20. In 1995, 
Cocchi et al. demonstrated that chemokine peptides, MTP-la, MTP-ip, RANTES, were 
the major HIV suppressive factors produced by CD8+ T cells. One year later, a G-protein 
coupled receptor, termed fusin, was characterized as an HTV-1 entry cofactor based on the 
fact that CD4+ cells made to express this receptor became susceptible to T cell-tropic 
HTV-1 infection (Feng et al., 1996). Fusin was identified later as the receptor for a unique 
CXC chemokine, SDF-1. 

Five independent groups published their work identifying a CC chemokine 
receptor, CCR5, as the co-receptor for Macrophage-tropic HTV-1 entry into CD4+ cells 
(Deng et al., 1996, Dragic et al., 1996, Alkhatib et al., 1996, Doranz et al. 1996, Choe et 



25 

al., 1996). Evidence from Choe et al. (1996) demonstrated that 1 ) CCR5, when expressed 
along with CD4, allowed cell lines resistant to most primary HTV-1 isolates to be 
infected, 2) CCR3 facilitated infection by a more restricted subset of primary virus, 3) 
eotaxin, one of the ligands for CCR3, inhibited infection by these isolates, and 4) the 
sequence of the third variable region of the HTV gpl20 exterior envelope glycoprotein is 
the important region for HTV-chemokine receptor interaction. Doranz et al. (1996) 
demonstrated that besides CCR5 and CXCR4, the receptors CCR3 and CCR2b are entry 
cofactors for a dual-tropic HTV-1 isolate (89.6). Later, V28 (human CX3CR1), and 
CCR8 were identified as coreceptors by certain strains of HTV-1 (Rucker et al., 1997, 
Combadiere et al., 1998, Jinno et al., 1998). 

The role of CCR5 as a cofactor for HTV-1 entry became unequivocal when Liu et 
al. (1996) identified two individuals that had been multiply exposed to HTV-1 but 
remained uninfected, had inherited a homozygous defective CCR5. The encoded 
defective CCR5 cannot be detected at the cell surface. Thus, These cells are resistant to 
HTV infection. 

In order to determine the important regions for the fusion property of CCR5, a 
number of studies using chimeras have been carried out. Rucker et al. (1996) determined 
regions in CCR5 and CCR2b that set HTV-1 tropic specificity by making CCR5/CCR2b 
chimeras. They found that residues 2-5 in the N-terminus of CCR5 are important in M- 
tropic virus fusion whereas residues 6-9 are important for dual-tropic (89.6) virus fusion. 
Atchison and colleagues (1996) demonstrated that various segments of extracellular 
regions of human CCR5 are sufficient to reconstitute fusion in the human CCR5/mouse 



26 

CCR5 chimera. In addition, data from studies of CCR5/CCR2 chimeras suggests that 
either the N-terminus or the rest of CCR5 are sufficient for M-tropic HTV fusion. 
Inhibition of HTV Entry by Chemokine Ligands: 

Since Cocchi et al. first reported the inhibitory effect of MUM a, MTP-ip, 
RANTES on HTV infection, there have been several studies examining the role of 
chemokine ligands in suppressing HTV infection. With the identification of MCP-2 as a 
ligand for CCR5, Gong et al. (1998) demonstrated that this peptide blocks CD4/CCR5- 
mediated HTV-1 entry/replication. Evidence from other groups proved the ability of 
inhibitors for either CCR5 or CXCR4 in blocking HTV-1 entry of the respective strain 
(Simmons et al., 1997, Howard et al., 1998). While the mechanisms by which 
chemokines and coreceptor inhibitors block HIV-1 infection are not fully elucidated, 
studies from Amara et al. (1997) and Howard et al. (1998) indicated that these molecules 
induced coreceptor internalization. 

Interactions between HIV and coreceptors have been shown to inhibit the binding 
between chemokine peptides and their receptors. Studies from Wu et al. ( 1 996) and 
Trkola et al. (1996) suggested that CD4 could induce the interaction between gpl20 
glycoprotein of HIV-1 and CCR5 resulting in inhibition of the binding of MlP-la and 
MIP-1 p to CCR5. On the other hand, studies carried out by Dolei et al. (1998) suggested 
that ligands for CCR5, MIP-1 a, MTP-ip, and RANTES, increased the replication of some 
T-cell-tropic HIV-1 strains and induced expression of the CXCR4 coreceptor. 

To further explore the effect of HTV envelope protein on target cells after binding 
to these chemokine receptors, Davis and colleague's (1997) work indicated that HTV-1 
envelope glycoproteins from both T-tropic and M-tropic strains activated a signal 



27 " 

tansduction pathway that induced tyrosine phosphorylation of the protein tyrosine kinase 
Pyk2 of CXCR4+ or CCR5+ target cells. These effects of HTV-1 on target cells through 
chemokine receptors are helpful for better understanding the pathogenesis of HTV-1. 
Chemokine Receptors and HTV in the CNS 

In the CNS, microglia are the principal target cells for HTV infection. Several 
groups examined the coreceptors that were responsible for HTV- 1 infection and got some 
contradictory data. He, et al. (1997) reported the expression of CCR3 and CCR5 in 
microglia of primary human brain cultures. Their data suggested that anti-CCR3 and 
anti-CCR5 antibody can both block M-tropic and brain-tropic HIV-1 isolates infection to 
these microglia, indicating that CCR3 and CCR5 are cofactors for some strains of HTV-1 
infection in microglia. Another study carried out by Shieh et al. (1998) suggested that 
predominantly CCR5, and to a much less extent CCR3 or CXCR4 are coreceptors for 
some of the HTV-1 isolates in cultured human microglia infection. However, while 
Ghorpade et al. confirmed the expression of CCR3 and CCR5 in cultured human 
microglia, their usage of antibodies to CCR3 or CCR5 failed to demonstrate similar 
levels of blocking of HTV-1 infection or replication. They concluded that there might be 
other chemokine receptors in microglia that are responsible for HIV-1 infection. 

Elucidation of the roles of chemokine receptors in HTV infection will be very 
useful in developing therapeutic regimes in AIDS treatment. 






28 
Specific Aims 

There is great interest in characterizing the expression, regulation, and functional 
roles of chemokine receptors in the CNS. Thus, the specific aims of this dissertation 
addressed three major areas. 

1 . The molecular cloning and functional characterization of rat CCR2, CCR5, and 
examination of the expression of these receptors in the CNS both in vitro (cultured 
microglia) and in vivo (EAE). 

2. Characterization of the expression of a chemokine:receptor pair, rat 
fractalkine:CX3CRl, in the CNS. Determination of expression site(s) of these 
molecules in the facial motor neuron regeneration model. 

3. Examination of expression of rat fractalkine and CX3CR1 in rat glioma models (RG2 
and C6 gliomas). 



29 




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Figure 1-2. Lectin (GSA I-B4) Staining of Facial Motor Nucleus Four Days After Facial 
Nerve Transection 

Microglia are stained by lectin from griffonia simplicifolia. Upper panel shows facial 
motor nucleus of the unoperated side and lower panel shows facial motor nucleus four 
days after facial nerve transection. Figure was kindly provided by Dr. Wolfgang J. Streit. 



CHAPTER 2 
MATERIALS AND METHODS 



Materials 

DNA modifying enzymes, RNA polymerases and the random primer labeling kit 
were purchased from Promega (Madison, WI) with the exception of DNA polymerase 
(pfu) which was purchased from Stratagene (La Jolla, CA). Reverse transcriptase, TRIzol 
Reagent, DMEM, trypsin, penicillin/streptomycin, LipofectAMTNE and rat recombinant 
interferon-y were from Life Technologies, Inc. (Grand Island, NY). In vitro transcription 
kit was purchased from Ambion (Austin, TX). [Alpha- 32 P]dCTP (3000 Ci/mmol) and 
[alpha- 33 P]dATP (2000 Ci/mmol) were from DuPont/NEN (Wilmington, DE). [Alpha- 
33 P]rUTP (2500 Ci/mmol) was from Amersham (Arlington Heights. IL). DNA 
sequencing kit (Sequenase, v. 2.0) was from U.S. Biochemical Corp. (Cleveland, OH). 
HEK293 cells were from ATCC (Rockville, MD). FURA2-A/M was from Molecular 
Probes (Eugene, OR). Xenopus laevis were purchased from Nasco (Fort Atkinson, WI). 
The mammalian expression vectors, pcDNA3 and pCLDN-lOb, were from Invitrogen 
(San Diego, CA) and SmithKline Beecham (King of Prussia, PA), respectively. Guinea 
pig myelin basic protein, complete Freund's adjuvant (CFA), lectin (GSA I-B4), and 
monoclonal-GFAP antibody were from Sigma (St. Louis, MO). Biotinylated anti-mouse 
IgG and horseradish peroxidase-avidin D were from Vector (Burlingame, CA). 



32 



33 

Mycobacterium tuberculosis (H37Ra) was from Difco (Detroit, MI). Male Lewis rats 
(200-250 g) and male Sprague Dawley rats (170-200) were obtained from Harlan 
(Indianapolis, EN). Murine MCP-1, murine MIP-loc, murine MIP-ip, human RANTES, 
rat fractalkine, human TGF-(5 were from R&D system (Minneapolis, MN). NTB2 
autoradiographic emulsion D19 developer and fixer were from Kodak (Rochester, NY). 
TdT transferase and biotin- 1 6-dUTP were from Boehringer Mannheim (Indianapolis, IN). 
An 829-base pair cDNA containing sequences encoding rat cyclophilin was kindly 
provided by Dr. A. John Maclennan (University of Florida, Gainesville, FL). Rat 
genomic DNA libraries, HaelH partial digest (lambda Charon 4A) was a kind gift of Dr. 
Kevin R Lynch (University of Virginia, Charlottesville. VA). 

Methods 

Polymerase Chain Reaction (PCR) Amplification of Novel DNA Sequences 

Two degenerate oligonucleotides 5'-TAC CTS CTS AAC CTG GCC HTL JSY 
GA-3' and 5'-ATD AQR GGP TTP ALP CAG CHG TG-3' (where Y=T or C, S=G or C, 
P=A or G. R=T or G, H=A/T, L=C/T/G, Q=T/C/A, J=A/T/G and D=A/G/C) were 
synthesized and used as primers for the amplification of DNA fragments (using rat 
genomic DNA as the template) encoding novel rat GCRs. The amino acid sequences 
encoded by these oligonucleotides correspond to highly conserved regions of G-protein 
coupled receptors (and specifically chemokine receptors). The conditions for the 
amplification of DNA in the polymerase chain reaction (PCR) were: 94 °C (1 minute), 
45°C (2 minutes), and 72°C (3 minutes), 30 cycles. PCR products were subcloned to the 



34 " 

Smal site of pGEM7Zf(+) (Promega) and subjected to DNA sequence analysis (Sanger 
dideoxy chain termination, Sequenase kit). 
Isolation of Rat Genomic Clones 

Approximately 1 x 10 6 independent recombinants of a HaelE partially digested 
Sprague Dawley rat genomic DNA library (lambda Charon 4A) was screened with the 
32 P-radiolabeled 0.7-kbp (pCrecl3) PCR products. Fifteen pCrecl3 hybridizing positive 
signals were plaque purified and phage DNAs purified by standard methods (Sambrook et 
al., 1989). Restriction-digested recombinant rat genomic DNAs hybridizing to the 
pCrecl3 DNA were subcloned to pGEM7Zf(+) and subjected to partial DNA sequence 
analysis (Sanger dideoxy chain termination, Sequenase kit). 
Southern Blot Analysis of Rat Genomic DNA 

Rat genomic DNA (15 ug, from normal adult Wistar rat liver) was restriction 
endonuclease-digested and subsequently electrophoresed through 0.85% agarose gel and 
subjected to Southern blot analysis (Sambrook et al., 1989). The transferred DNAs were 
hybridized to the 32 P-radiolabeled 1.0 kbp Clal/BamHl fragment of the rat CCR5 gene 
using the method of Church and Gilbert (1984). Final wash temperatures were 50°C and 
65°C. 
Transfection of HEK293 Cells: 

The open reading frame (orf) of rat CCR2 was amplified using the PCR and the 
cloned genomic DNA as the template. Two oligonucleotides were synthesized and used 
in the PCR (30 cycled of 94°C for 1 minute, 55°C for 2 minutes, and 72°C for 3 minutes). 
The oligonucleotides used were 5'-TTC TCT GTC CAC AGA ATC and 5'-CTA CTT 






35 



ACT TTA CAA CCC. The PCR product was reacted with the Klenow fragment of DNA 
poll in the presence of dNTPs and cloned subsequently to the EcoRV site of pcDNA3. 
The resultant expression plasmid construct was subject to DNA sequence analysis to 
verify the fidelity of the Pfu DNA polymerase used in the PCR. The Dral/BamHI 
fragment containing the rat CCR5 orf was cloned to the Hindm and BamHI sites of 
pCLDN-lOb; the HindUl site was made blunt by the action of the Klenow fragment of 
DNA poll in the presence of dNTPs. A KpnI/BamHI fragment of this pCLDN-lOb 
construct was subcloned subsequently to the same sites of pcDNA3. HEK293 cells were 
grown in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS) and 
penicillin/streptomycin (complete media). HEK293 cells (2 x 10 3 cells) were transfected 
with lOug of plasmid DNA using LipofectAMTNE reagent according to the 
manufacturer's recommended procedure. Approximately 72 hours post transfection, cells 
were replaced in complete media containing the neomycin analog G41 8 (0.8 mg/ml). 
Clonal cells resistant to G418 were isolated and propagated for further analysis. Cells 
expressing (c)RNA hybridizing to G418 were isolated and propagated for further 
analysis. Cells expressing (c)RNA hybridizing to the Clal/BamHI DNA fragment of rat 
CCR5 were characterized further. 
Measurement of Intracellular Calcium Levels 

HEK293 cells (~5xl 6 ) which stably express rat CCR2 were dissociated from the 
culture dish and diluted subsequently into 10 ml MEM (serum- and antibiotic-free). The 
suspension was made in 5 mM of FURA-2/AM and was incubated for 30 minutes in the 
dark at 37°C. The cells were then washed twice with PBS buffer and subsequently 
resuspended in 2 ml HBSS buffer. The cell suspension was transferred to cuvettes and 



36 

placed in a SLM 8000 model spectrofluorimeter (driven by an IBM PC). FURA-2 

fluorescence was monitored at 510nm, with automatic slewing between excitation 

wavelengths at 340nm and 380nm. Ratios at these two excitations were automatically 

calculated. 

Expression of Rat CCR5 in Xenopus Laevis Oocytes 

Plasmid (pcDNA3) constructs containing the open reading frames of rat CCR2 
and rat CCR5 were linearized, and complementary RNAs were synthesized in an in vitro 
transcription reaction using T7 RNA polymerase. The transcription products were 
confirmed by Northern blot analysis. Xenopus laevis oocytes were injected with 50 ng of 
RNA in a total volume of 25 nl per oocyte. Electrophysiological recordings were made 3- 
4 days after injection in oocytes superfused with Oocytes Ringer's solution at room 
temperature under voltage-clamped conditions using two microelectrodes (both filled 
with 3 M KC1), with the membrane potential routinely clamped at -50 mV using a TEV- 
200 voltage clamp instrument. Test chemokines were dissolved in distilled water and 
diluted to a final concentration of 250 nM in Oocytes Ringer's solution. Thirty pi of each 
chemokine was applied directly onto voltage-clamped oocytes for 120s, and the current 
induced was monitored on a Tektronix 5113 dual beam storage oscilloscope linked to an 
IBM-PC. 
RNA Isolation, RNase Protection Assay, and Northern Analysis 

Total RNA was isolated using TRIzol Reagent according to the manufacturer's 
recommended procedure. The RNase protection assay was performed as previously 
described (Feng et al., 1994). The antisense riboprobe for rat CCR2 was generated from 
an in vitro transcription reaction using a plasmid construct containing the Dral/EcoRI 



37 

fragment (365-base pair) of rat CCR2. The antisense probe for rat CCR5 was made in a 
similar manner using an EcoRV/Bgin restriction fragment (3 10-base pair) of rat CCR5. 
The antisense probe for rat fractalkine and RBS 1 1 were made using total RNA (five ug) 
and J "P-UTP-labeled riboprobes generated from in vitro transcription reactions utilizing 
plasmid construct templates containing either a 0.4 kbp Ncol fragment of rat fractalkine 
cDNA or a 0.45 kbp Smal/Kpnl fragment of RBS 1 1 cDNA. 

Total RNA (10 ug/lane) extracted from various cell and tissue preparations was 
electrophoresed through denaturing 1.2% agarose and subjected to Northern blot analysis 
(Church and Gilbert, 1984). For hybridization probes, the following DNAs were used: 
the 1.5-kbp Clal/BamHI fragment of the rat CCR5 gene, a 1.1 -kbp RBS1 1 protein coding 
DNA sequence, a 700 base pair cDNA fragment for rat fractalkine, and an 829-base pair 
cDNA encoding rat cyclophilin, all of which were 32 P-radiolabeled by the random primer 
method (1-2 x 10 9 dpm/ug). 
Preparation of Primary Cultures of Rat Microglia 

Microglial cultures were prepared as previously described, with minor 
modifications (Giulian and Baker, 1986). In brief, brains were removed from neonatal 
rats and placed in an isotonic salt solution containing 100 U of penicillin G, 100 ug of 
streptomycin and 0.25 ug of amphotericin B (Fungizone) per ml (pH 7.4). All blood 
vessels and pia mater were removed, and the diencephalon were dissected out and 
chopped into approximately 2 to 3 mm chunks. The brain pieces were then dissociated 
by trituration at 37°C in 20 ml of isotonic saline solution (pH 7.4) containing 0.25% 
trypsin (wt/vol) and DNase-I (8 ug/ml). The dissociated cell suspension was added to an 
equal volume of DMEM containing 10% FBS; the suspension was then centrifuged at 



38 

1000 x g for 10 min and the resulting pellet washed with 50 ml DMEM containing 10% 
FBS. Cells were resuspended in DMEM containing 10% FBS to a density of 1 x 10 5 
cells/ml and 10 ml of this suspension were added to 100 mm poly-L-lysine precoated 
culture dishes and incubated at 37°C in a humidified incubator with 5% C02/95% air. 
On day 3, the culture medium was replaced with fresh DMEM containing 10% FBS. 
After 1 1 aditional days of incubation of the mixed primary astroglial/microglial cultures, 
the flasks were rotary-shaken for one hrs (100 rpm, 37°C) and the culture media, which 
contained dislodged free-floating microglia, was removed and centrifuged at 1000 x g for 
1 5 min. The resulting microglial pellet was resuspended in DMEM and plated onto 100 
mm dishes at a density of 5 x 10 6 cells per dish. Microglia were cultured for 
approximately 24-48 hours prior to treatment. 
Induction of Rat EAE: 

Male Lewis rats (-200 - 250 g) were injected in the hind foot pad, under 
methoxyflurane anesthesia, with an emulsion (0.1 ml) of 125 ug purified guinea pig 
myelin basic protein (MBP) in Complete Freund's Adjuvant (CFA. Difco) supplemented 
with 50 ug Mycobacterium tuberculosis (H37Ra). Clinical disease, i.e. various degrees 
of paraparesis, was typically evident between days 10 and 12 post injection. Animals 
(EAE and control) were screened for neurological signs on a daily basis according to the 
following scoring system: 0: no signs; 1 : flaccid tail, general depression of activity; 2: 
mild paraparesis, unsteady gait; 3: moderate paraparesis, legs splayed apart but voluntary 
movements of affected extremities possible; 4: complete paraparesis, extremities without 
voluntary movements; 5: death due to EAE. If the clinical sign remained in one stage for 
2-3 days or reached stage 4, the rat was perfused intracardially with PBS and the lumbar 



39 

enlargements of the spinal cords removed. Total RNA was isolated and subsequently 
subjected to either RNase protection assay or Northern blot analysis as described above. 
Molecular Cloning and Sequencing of Rat Fractalkine cDNA 

A rat brain cDNA library (Strategene, CA) was screened by hybridization using a 
rat factalkine cDNA probe. The hybridization probe was generated by PCR using primers 
derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine 
cDNA was sequenced by standard methods (Sanger dideoxy chain termination, 
Sequenase kit). 

An EcoRI/Sall DNA fragment containing sequences encoding full length rat 
fractalkine protein was subcloned to the mammalian cell expression vector, pCDM8 
(Invitrogen). DNA containing human fractalkine protein coding sequence was generated 
by PCR and subcloned into the same vector. To generate peptidase-resistant membrane- 
anchored fractalkine, both human and rat fractalkine mutant forms were also prepared by 
PCR, in which the putative dipeptidase site Arg-Arg, adjacent to the transmembrane 
spanning domain, was mutated into Ser-Ala. 
Expression and Functional Characterization of Rat CX3CR1 in Transfected Cells 

A cDNA containing the open reading frame of RBS1 1 was cloned to the EcoRV 
site of pcLDN-lOb. CHO cells (grown in Ham's F12/10% heat inactivated fetal bovine 
serum (FBS)/penicillin/streptomycin) were transfected with 10 ug of plasmid DNA using 
LipofectAMINE reagent (Life Technologies, Inc.). G4 1 8-resistant clonal cells expressing 
(c)RNA hybridizing to RBS 1 1 cDNA were characterized further in binding and 
functional assays. HEK293T cells (grown in DMEM/10% FBS/pen/strep) were 



40 

transfected using LipofectAMNE and 10 ug of rat or human fractalkine encoded 
DNA/100 mm plate and used three days post-transfection. 

Synthetic chemokines were generated by native chemical ligation of peptides 
synthesized by solid-phase methods (Applied Biosystems 430A Peptide Synthesizer), 
purified by reverse-phase high-performance liquid chromatography, and characterized by 
electrospray mass spectrometry (Dawson et al., 1994). Purified synthetic chemokines 
were reconstituted in phosphate buffered saline (PBS) and stored frozen, until use, at 
minus 70°C. 

Whole cell binding analysis was performed on three million PBS-rinsed cells per 
35 mm well. Cells were incubated (1 hour at room temperature) in 1 ml HBSS/0.1% 
BSA, I-labeled human fractalkine/chemokine domain (100 Ci/mmol) and various 
concentrations of cold competing chemokine ligands. At the end of the incubation, the 
cells were rinsed 3 x 2 ml with ice-cold HBSS/0.1% BSA. Total radioactivity retained in 
the wells was removed with 0.2 N NaOH and quantitated by gamma spectroscopy. 
In Situ Hybridization Analysis 

In situ hybridization was carried out according to previously published procedures 
(McNamara and Routtenberg, 1995). Radiolabled [ 33 P] (c)RNA probes were prepared by 
in vitro transcription from T7 or Sp6 RNA polymerase promoters in the pGEM7Zf(+) 
plasmid system. For RBS1 1 probes, a full length cDNA clone in pGEM7Zf(+) was 
digested with Kpni and a resultant 450 bp fragment was isolated and subcloned to the 
Smal site of pGEM7Zf(+). The antisense probe for RBS1 1 was prepared by linearizing 
the pGEM7Zf(+) plasmid with HindlTJ. For preparation of the sense strand probe for 
RBS1 1 the plasmid was digested with EcoRI. For rat fractalkine probes, a cDNA clone 



41 

encoding chemokine domain of rat fractalkine was digested with Ncol. An 
approximately 400 bp fragment was purified and subcloned to the Smal site of 
pGEM7Zf(+). Antisense probe was generated by digesting this construct with EcoRI and 
sense strand probe was obtained with BamHI digestion. In vitro transcription and 
labeling of RNA probes was performed as described (McNamara and Routtenberg, 1995) 
with mild modifications. Briefly, lOOuCi of [alpha- 33 P]-rUTP was added in a total 10 ul 
reaction [40 mM Tris; 6 mM MgC12, 2 mM spermidine; 10 raM NaCl; 4 mM 
dithiothreitol; 0.8 U/ml RNase inhibitor; 0.5 mM each ATP, CTP, GTP; linearized 
template equivalent to 0.5 ug of whole plasmid; and 10 U of either T7 or Sp6 RNA 
polymerase]. The reaction was allowed to proceed for 60 minutes at 37°C. The DNA 
template was removed by RNase-free DNase (10 U) for 15 minutes at 37°C. Proteins and 
free ribonucleotides were removed by NH4Ac and ethanol precipitation. Brain tissues 
were embedded in O.C.T. compound (Miles Inc., Elkhart, EN) and 12-20 urn thick frozen 
sections were made and placed on Superfrost/Plus microscope slides (Fisher Scientific, 
Pittsburgh, PA). After fixed in 4% paraformaldehyde buffered PBS, pH 7.4. for 10 min 
in 4°C, tissue sections were washed in DEPC treated water and acetylated in a freshly 
prepared solution of 0.1 M triethanolamine with acetic anhydride, pH 8.0, for 10 minutes. 
After washing in 2x SSC, sections were dehydrated through graded ethanols. Sixty ul of 
the hybridization mixture (50% formamide, 0.3 M NaCl, 20 mM Tris, pH 8.0, 5 mM 
EDTA, 1 x Denhardt's, 10% Dextran Sulfate, 10 mM DTT) containing 1.5x 10 6 cpm 
counts of radiolabeled RNA probe was applied to each slid and covered with a siliconized 
coverslip. Hybridization was carried out for 16-18 hours at 60-65°C in a humidified 
chamber. The excess radioactivity was removed by washing the slides with two 20-min 



42 ' ' 

rinses of standard saline citrate (SSC) buffer (0.3 M NaCl, 30 mM sodium citrate), and a 
30-min digestion in RNAse A at 37°C in buffer containing 500 mM NaCl, 10 mM Tris, 
pH 8.0, and 1 mM EDTA. After two more rinses with SSC, sections were stringently 
washed in 0.1 X SSC at 60°C for two hours, followed by two 10-min rinses in 0.5X SSC 
at room temperature and dehydrated in graded ethanols containing 300 mM ammonium 
acetate. Sections hybridized with "sense" radiolabeled riboprobe served as a control. 
Lectin Staining and Immunohistochemical Analysis 

Following in situ hybridization and prior to emulsion autoradiography, some 
sections were stained with either isolectin from Griffonia simplicifolia (GSA I-B4, to 
identify microglia) according to previously published procedures (Streit, 1990) or with 
anti-GFAP (glial fibrillary acidic protein, to identify astrocytes) in order to determine 
cellular localization of RBS1 1 or fractalkine. 

For lectin staining, sections were soaked in PBSC (PBS containing 0.3% Triton 
X-100 and 0.1 mM CaCl 2 , MgCl 2 , and MnCl 2 ) at 4°C for 1 hour before being incubated 
with GS I-B4-HRP (10 ug/ml) in PBSC overnight at 4°C. The sections were briefly 
rinsed with PBS and the lectin binding sites were visualized using 3,3' - 
diaminobenzidine-H202. 

For GFAP staining, sections were rehydrated in PBS for 10 minutes and blocked 
with PBS/10% goat sera for 30minutes before incubating overnight in 4°C with anti- 
GFAP antibody diluted 1 : 1000 in PBS/10% goat sera. After washing in PBS 3x 10 
minutes, sections were then incubated with biotinylated anti-mouse-IgG (from goat) for 
45 minutes in room temperature. With another extensive wash using PBS, 3x 10 



43 

minutes, sections were treated with horseradish peroxidase avidin D for 45-60 minutes 
and rinsed with PBS before exposed to 3,3' -diaminobenzidine-H20 2 . 

Sections were soaked in PBS for 5 minutes and allowed to air dry. The slides 
were then exposed to films for 24 hrs and developed in D19 developer for 5 min, rinsed 
in water 5min, and fixed for 8 min. If the films showed that the hybridization was 
successful, the sections were exposed to Kodak NTB2 autoradiographic emulsion at 4°C 
for a period of time depending on the intensity of the signals on the films generally 
between 2-6 weeks and then developed in D19 developer for 2 minutes, rinsed in tap 
water for 5 minutes, and fixed for 5 minutes in fixer. After washing in tap water, the 
slides were counterstained in cresyl violet or hematoxylin, dehydrated in ascending 
ethanols and coverslipped with Permount. 
Facial Nerve Transections 

Facial motor nerve transections on methoxyflurane-anesthetized male Sprague- 
Dawley rats (175 - 200 g) were carried out according to previously published procedures 
(Streit et al., 1988). In brief, Male Sprague-Dawley rats (175-200 g) were anesthetized 
with methoxyflurane. The right facial nerve is exposed and transected at the level of the 
stylomastoid foramen. The unoperated contralateral side serves as the control. Following 
1, 4, 7, 14, and 21 days after axotomy, rats (n = 3 per time point) were sacrificed with an 
overdose of sodium pentobarbital (75 mg/kg) and perfused transcardially with lx PBS 
(pH 7.0) followed by 4% paraformaldehyde/0.1 M P04 buffer (pH 7.4). Brains were 
saturated in 30% sucrose/0.1 M P04 buffer (pH 7.2) and frozen in 2-methylbutane 
(chilled to -80oC before use), and then stored at -80°C. At a later time, the brain slices 



44 

were prepared for in situ hybridization and immunohistochemistry analysis as described 
above. 

Quantitation of areal grain densities of bilateral facial motor nuclei was performed 
on emulsion-dipped slides under dark field conditions using a video imaging system 
(Microcomputer Imaging Device) and image analysis program (Imaging Research Inc.) 
interfaced via a video camera (Sony XC77) connected to a Zeiss microscope (Axioscop 
20). Statistical comparisons (one-way analysis of variance) were made between 
ipsilateral and contralateral values within each post-lesion time point. 
Tumor Inoculation 

The RG-2 or C6 glioma cell lines were maintained in a monolayer culture in 
DMEM with 10% FBS. Female Fisher rats were anesthetized with sodium pentobarbital. 
Tumor cells (5xl0 5 /5ul) were injected stereotactically into the right cerebral hemisphere 
through a burr hole. Five ul of saline solution was injected into a group of rats that 
served as controls. 
Terminal Transferase-Mediated UTP Nick End Labeling (TUNEL-Labeling) 

TUNEL-labeling was performed following the protocols by Gavrieli and 
colleagues (1992) with some adjustments. RG2 or C6 cell inoculated Fisher rats were 
sacrificed, fixed and cryoprotected as described for in situ hybridization analysis. After 
rinse in water 2x5 minutes, endogenous peroxidase was inactivated by incubating the 
slides in 3% water for 10 minutes at room temperature. Tissues were then exposed to 
0.2% triton X-100/PBS for 5 minutes in room temperature. Slides were incubated in 
water at 60°C for one hour and cooled down in room temprature before TdT treatment. 
After pre-incubating in TdT buffer (30 mM Tris-HCl, pH 7.2, 140 mM sodium 



45 

cacodylate, 1 mM cobalt chloride) for 5 minutes, the tissue sections were incubated in 
TdT buffer containing 2.5 U/ul terminal transferase and 10 umol biotinylated 16-dUTP in 
a humid atmosphere for 1 hour at 37°C. The reaction was stopped by transferring the 
slides into 300 mM sodium chloride, 30 mM sodium citrate for 20 minutes in room 
temperature. After blocking the coverslips for 30 minutes in 2% bovine serum albumin 
(BSA), they were incubated with the ABC kit for 30 minutes at room temperature and 
developed with 3,3'-diaminobenzidine as substrate. Negative controls were carried out 
by omission of terminal transferase and biotinylated UTP. 



CHAPTER 3 

CHEMOKINE RECEPTOR EXPRESSION IN CULTURED GLIA AND RAT 

EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS 



Introduction 

Chemokines are a growing family of peptides that are involved in leukocyte 
recruitment and activation. These peptides can be divided into four subfamilies (CC, 
CXC. C, and CX3C subfamilies) based on the relative positions of highly conserved 
cysteine residues in their amino acid sequences. 

Some of the receptors for chemokines have been identified by molecular cloning 
techniques and they are members of the rhodopsin superfamily of G-protein coupled 
receptors (GCRs). These receptors are characterized by their seven transmembrane 
spanning helices in their amino acid sequences. Although most chemokine receptors 
interact with more than one chemokine peptides, in general, they can only be stimulated 
by chemokines from the same subfamily. To date, the primary structures of at least 
sixteen functional human chemokine receptors have been determined. Five of these 
receptors bind, and are activated by, C-X-C chemokines nine receptors are activated by 
C-C chemokine peptides, one receptor binds the C chemokine lymphotactin and one 
receptor is bound and activated by the CX3C chemokine fractalkine. Genes encoding 
chemokine receptors have also been identified in the mouse and rat. It has been 
demonstrated that human CXCR4, CCR5, CCR2, CCR3, CCR8, and CX3CR1 act as 
cofactors for entry into cells of primary isolates of HTV. While the expression and 

46 



47 

functions of chemokines and chemokine receptors have been extensively studied in the 
periphery, less information, regarding these genes, is available in the central nervous 
system. 

Microglia, as the immune responsive cells in the CNS, comprise 5-20% of the 
total glial cell population (Graeber et al., 1990, Kreutzberg 1996). These cells react to 
even minor pathological changes in the CNS and serve as a key factor in the defense of 
the neural parenchyma against infectious agents, inflammation, trauma, ischemia, brain 
tumors and neurodegeneration. Several chemokines and chemokine receptors have been 
shown to be expressed in microglia including MCP-1, MP- la, MTP- 1 (3 (McManus et al., 
1998), RANTES (Miyagishi et al., 1997), SDF-p (Ohtani et al., 1998), IP- 10 (Ren et al., 
1998), CXCR4 (Lavi et al., 1997), CCR3, and CCR5 (Xia et al., 1998). 

Microglia activation and proliferation occurs in experimental allergic 
encephalomyelitis (EAE). EAE is a cell-mediated autoimmune disease of the central 
nervous system induced by a number of different antigen preparations including myelin 
basic protein. Its histopathology is characterized by perivascular mononuclear cell 
infiltration and microglia and astrocyte activation. Clinical signs of EAE consist of 
hindlimb and tail paresis as well as paralysis. Since it has many clinical and pathological 
features in common with multiple sclerosis, it is considered to be an animal model of this 
disease. Several groups have demonstrated that a number of chemokine peptide were 
expressed or up regulated in the spinal cords of EAE animals. These chemokines include 
RANTES, MP- la, MP- 113, TCA3, IP- 10, MCP-1, KC/GRO, MCP-3, SDF, and Mff-2 
(see chapter one). 



48 

Towards our goal of defining chemokine receptor expression in the CNS, I have 
isolated, by molecular cloning techniques, rat genes encoding the chemokine receptors 
CCR2 and CCR5. I describe expression patterns of these genes in cells and tissues of the 
CNS and report regulation of chemokine receptor expression in vitro in cultured rat 
microglia and demonstrate in vivo modulation of chemokine receptor expression in EAE 
rats. 

Results 

Isolation of Rat Genomic DNA Containing Sequences Encoding Novel G-Protein 
Coupled Receptors 

Degenerate oligonucleotide primers were designed based on the highly conserved 
regions (TM2, TM3, and TM7) of the known human chemokine receptors (huEL-8RA, 
huIL-8RB, huCCRl, and huCCR2 receptors) and the orphan rat chemokine receptor-like 
gene, RBS1 1 (Holmes et al., 1991; Murphy et al., 1991; Neote et al., 1993; Gao et al., 
1993; Charo et al., 1994; Harrison et al., 1994). DNA fragments encoding novel rat 
chemokine receptors were generated by PCR reactions using these oligonucleotides and 
rat genomic DNA as the template. Consequently, these PCR products were utilized for 
further screening of a Sprague Dawley rat genomic library under high stringency 
conditions in order to isolate the full length DNA sequenses. Genomic library screening 
using one of the fragments, pCrecB, resulted in 15 plaque pure bacteriophage clones. 
Restriction endonuclease digestion and DNA sequence analysis of each of these clones 
revealed two distinct sets of bacteriophage containing open reading frames (orfs) 
encoding novel G-protein coupled receptors with high sequence identity to other known 
chemokine receptors. Hereafter, we designate the orfs containing the pCrecl3 sequences 



49 

as rat CCR2 and rat CCR5. Figure 3-1 depicts a multiple alignment of rat, human and 
murine CCR2 and CCR5. Rat CCR2 encodes a protein of 373 amino acids with a 
calculated molecular mass of 42,763 Da, while rat CCR5 is a protein of 354 amino acids 
and a calculated molecular mass of 41,017 Da. Both receptors contain structural 
characteristics of CC chemokine receptors. There is a preponderance of acidic amino 
acid residues in their amino acid sequences of the respective N-termini. Putative N- 
linked glycosylation sites (N-X-S/T) can also be identified. The C-termini of each of 
these rat receptors are serine rich, suggesting possible receptor regulation by 
phosphorylation. 

A comparison of amino acid identities of the rodent, murine and human receptors 
is shown in Table 3-1 . Rat CCR2 is 79% and 93% identical to human and murine CCR2 
respectively while rat CCR5 is 83% and 92% identical to human and murine CCR5, 
respectively. The most variable regions between rat and human orthologs were found in 
putative extracellular portions (N-termini and loops). The high amino acid identity 
between rat and human or murine CC chemokine receptors suggested the identity of these 
novel rat chemokine receptors and allowed us to designate these rat receptors according 
to nomenclature standardized for the chemokine receptor family. 
Signal Transduction Properties of Rat CCR2 and CCR5 

In order to confirm the identities of rat CCR2 and CCR5, functional assays were 
carried out on cells expressing each of these receptor genes. Application of murine MCP- 
1 stimulated a transient elevation in intracellular calcium ([Ca 2+ ],) levels in rat CCR2 
expressing HEK293 cells (Fig. 3-2) in a dose dose-dependent manner. The EC50 value 
for this effect is approximately 0.4 nM. However, rat CCR5 expressing HEK293 cells 



50 

did not show a detectable change in [Ca 2+ ]j in the presence of either murine MCP-1 or 
MlP-la. Electrophysiological experiments were carried out to examine the calcium 
mobilization of CCR5 expressing Xenopus laevis oocytes in response to ligand 
application. Figure 3-3 demonstrates traces of Ca 2+ -activated CI" current recorded from 
Xenopus laevis oocytes microinjected with CCR5 (c)RNA and exposed to MlP-la or 
MIP-1B. No response can be detected when the cells were exposed to the buffer only 
(data not shown). 
Genomic Analysis 

Southern blot analysis of restriction-digested rat genomic DNA probed with 
radiolabeled DNA encoding rat CCR5 showed consistent hybridization to two distinct 
sets of genomic fragments (Fig. 3-4). These hybridizing restriction fragments were of 
identical size to the hybridizing restriction fragments from the isolated rat genomic 
bacteriophage clones. These two sets of the hybridizing signals can be seen even under 
more stringent wash conditions as shown in the left panel of Figure 3-4 where the blot 
was washed at 65°C as compared to the right panel with the wash temperature at 50°C. 
Tissue Specific Expression of Rat CCR2 and CCR5 

The tissue and cell distribution of rat CCR2 and CCR5 mRNAs were determined 
by RNase protection assay. Analysis of total RNA from solid rat organs and cells probed 
with an antisense riboprobe of rat CCR2 revealed that adult rat lung, spleen, kidney, 
thymus, and macrophages are sources of CCR2 (Fig. 3-5). CCR2 mRNA was not 
detected in rat liver or rat brain (data not shown). Similar to that of rat CCR2, rat CCR5 
mRNA was also found to be expressed in rat lung, spleen, kidney, thymus, and 



51 

macrophages (Fig. 3-6). However, in addition, CCR5 mRNA was detected in extracts of 

whole rat brain. 

Expression and Regulation of the Rat CCR2 and CCR5 in Cultured Microglia 

The expression and regulation of the CCR2 and CCR5 gene in the cultured rat 
microglia was investigated by Northern blot analysis shown in Figure 3-7. RNA from 
microglial cells treated with various concentrations of IFN-y was isolated and a Northern 
blot was generated as described in chapter 2. This blot was subsequently probed with a 
1.5 kbp Clal/BamHI fragment of rat CCR5. Steady state levels of CCR5 mRNA present 
in the cultured microglia were dose-dependently increased by IFN-y treatment. CCR2 
mRNA was not detected in primary cultures of rat microglia (either control or IFN-y- 
treated, Figure 3-5). 
Modulation in Rat EAE 

A number of cytokines, including IFN-y and chemokine peptides, are known to be 
elevated in spinal cords from EAE animals (Merrill et al., 1992; Weinberg et al., 1993; 
Hulkower et al., 1993; Ransohoff et al., 1993; Godiska et al., 1995; Miyagashi et al., 
1995; Berman et al., 1996; Glabinski et al., 1997). In order to examine the expression of 
the receptors for chemokine peptides in vivo, in collaboration with Dr. Wolfgang J. Streit, 
we examined for the expression of chemokine receptor genes in rat EAE. EAE was 
induced in Lewis rats by hind footpad injection of myelin basic protein. Total RNA from 
the lumbar enlargements of the spinal cords from animals at various stages of the disease 
were examined for the presence of chemokine receptor mRNAs. Figure 3-8 depicts an 
analysis of multiple chemokine receptor (CCR2, CCR5, CXCR4, and RBS1 1) mRNA 



52 

levels in the lumbar enlargement of the EAE rats, as assessed by either RNase protection 
analysis or Northern blot analysis. For each receptor, increases in the levels of mRNA 
were observed in animals displaying clinical symptoms of the disease. In animals that did 
not display symptoms, either no increases in mRNA (as for CCR2, CCR5, and CXCR4) 
or minimal increases (as for RBS1 1) were seen. A correlation between the increase in 
receptor RNA and the degree of clinical symptoms was observed for CCR2 and CCR5, 
i.e. a greater increase in mRNA for these receptors was evident in the animal displaying 
complete hind limb paralysis. 

Discussion 

In the past few years, many research groups have carried out studies to determine 
the expression of chemokine peptides and chemokine receptors in the CNS. This project 
launched our series of studies in an ongoing effort to characterize the expression, 
regulation and functional roles of chemokine peptides and receptors in the central nervous 
system. Our results demonstrate that the rat genome contains orthologous genes encoding 
the chemokine receptors CCR2 and CCR5. Unlike rat CCR2, which is not constitutively 
expressed in the brain or cultured microglia, rat CCR5 mRNA is constitutively expressed 
in these tissues. In addition, steady state levels of chemokine receptor mRNA(s) are 
regulated both in vitro in the cultured microglia and in vivo in rat EAE. 

Two very closely related CC chemokine receptor genes, rat CCR2 and CCR5 have 
been identified via sequence homology techniques. The deduced amino acid sequences 
contain putative seven hydrophobic transmembrane alpha helices, which confirmed that 
they are members of the G-protein coupled receptor superfamily. Similar to many other 



53 

chemokine receptors (Ahuja and Murphy, 1993), there is a preponderance of acidic amino 
acid residues in the conceptualized amino acid sequences of the respective N-termini, a 
region that is likely to participate in binding with the positively charged N-termini of 
chemokine peptides. A serine rich region is found in the carboxy-terminal domain of 
both receptors, suggestive of potential phosphorylation regulation of these proteins. The 
amino acid sequences also carry potential N-linked glycosylation sites (N-X-S/T) for both 
receptors. The overall amino acid sequence identity between human and rat CCR2 and 
CCR5 are 79% and 83% respectively. The high amino acid sequence identity prompt us 
to hypothesize that these genes encode rat orthologs of the two human chemokine 
receptors. 

Functional analysis of these rat chemokine receptors in receptor-expressing-cell 
systems finalized their identity as rat CCR2 (MCP-1 receptor) and CCR5 (receptor for 
MlP-la, Mff-lp and RANTES), respectively. Similar to its human ortholog, rat CCR2 
can be stimulated by MCP-1 in low nanomolar concentrations. Although we failed to 
demonstrate a transient intracellular Ca 2+ increase in rat CCR5 expressing HEK cells, we 
were able to detect a Ca 2+ activated CI* current in rat CCR5 expressing oocytes in 
response to murine MOM a, MIP-ip and human RANTES. The identical ligand 
specificity between rat and human orthologs indicates that the rat can serve as a good 
model in studying the functional roles of human CCR2 and CCR5 under physiological 
and pathological circumstances. 

Rat CCR2 and CCR5 are found in a number of cells and tissues that can either 
produce or respond to chemokine peptides. In addition to the peripheral cells and tissues 
that express both rat CCR2 and CCR5, rat CCR5 is also constitutively expressed in the 



54 

brain as well as cultured microglia. This observation is in agreement with the finding that 
the human gene is expressed in human microglia present in primary brain cultures (He et 
al., 1997). In vivo studies will be necessary to prove unequivocally that rat CCR5 is 
expressed in microglial cells in the CNS. 

My data revealed that steady state levels of rat chemokine receptor mRNA were 
regulated in cultured microglia in vitro as well as in an EAE animal model in vivo. After 
IFN-y treatment, the expression of rat CCR5 mRNA is up-regulated in cultured microglia. 
On the other hand, the lesion of EAE involves in infiltration of the IFN-y producing T 
lymphocytes due to the break down of the blood brain barrier (Merrill et al., 1992, 
Weinberg et al., 1993). Taken together, it is conceivable that in normal rat spinal cord, 
the CCR5 hybridizing signal was contributed by endogenous microglia. The microglial 
expression of CCR5 is promoted by these EFN-y producing T lymphocytes during EAE. 
This hypothesis is supported by my data demonstrating the increased expression of CCR5 
mRNA in the spinal cords of EAE rats. While constitutive expression of CCR2 is absent 
in rat spinal cord, its expression is detectable in EAE rats. It is known that infiltrating 
macrophages are also found in the spinal cords of EAE rats (Huitinga et al., 1995). 
Cultured macrophages are capable of expressing CCR2, CCR5 and another chemokine 
receptor gene, RBS1 1, in vitro (Combadiere et al., 1995b; Raport et al., 1995). Thus, 
macrophages are likely to be the sources of CCR2 hybridizing signal and part of the 
increased CCR5 and RBS1 1 hybridizing signals in the spinal cords of diseased rats. 
Studies combining in situ hybridization and histochemical analysis should clarify the cell 
type expression of each of these receptors in the EAE model. 



55 ' 

The expression of chemokine receptor mRNA in the brain and spinal cord as well 
as cultured microglia indicates that these molecules may play some functional roles in the 
CNS under neurophathological and neurophysiological conditions. It is known that a 
number of chemokine peptide genes are induced in EAE, including MCP-1, MCP-3, 
MEM a, MP- IB, RANTES, TCA3, KC/CINC, and IP- 10 (Hulkower et al., 1993; 
Ransohoff et al., 1993; Godiska et al., 1995; Miyagashi et al., 1995; Berman et al., 1996; 
Glabinski et al., 1997). For some of these peptides, astrocytes have been identified as the 
cellular source (Ransohoff et al., 1993; Berman et al., 1996; Glabinski et al., 1997), while 
for others, infiltrating leukocytes were determined to be the source (Glabinski et al., 
1997). Our data demonstrated the presence of receptors of some of these chemokine 
peptides in the CNS, particularly in spinal cord of EAE rats. These data suggest a 
potential interaction between these chemokine peptides and their receptors in this 
neuropathological model of human multiple sclerosis which may play some functional 
roles during the disease process. Our identification of the rat genes encoding chemokine 
receptors CCR2 and CCR5 should enhance fundamental studies of these receptors in 
normal and pathological states of the CNS. 



56 



1 60 

r»tCCR5 MDFQGSI PTYIYDIDYS MSAPCQKVNV KQIAAQLLPP 

murCCR5 HDFQGSV PTYSYDIDYG MSAPCQKINV KQIAAQLLPP 

huCCR5 MDYQVSS P . . IYDINYY TSEPCQKINV KQIAARLLPP 

ratCCR2 KEOSNMLPQF IHGILSTSHS LFPRSIQELD EGATTPYDYD DGEPCHKTSV KQIGAWILPP 

BurCCR2 MEDNNMLPQF IHGILSTSHS LFTRSIQELD EGATTPYDHD DGEPCHKTSV KQIGAWILPP 

huCCR2b MLSTSRS RFIRNTNSSG EEVTTFFDYD YGAPCHKFDV KQIGAQLLEE 



ratCCR5 


61 

LYSLVFIFGF VGNMMVTLIL 


ISCKKLKSMT 


DIYLFNLAIS 


120 
DLLFLLTLPF NAHYAANENV 


murccrS 


LYSLVFIFGF VGNMMVFLIL 


ISCKKLKSVT 


DIYLLNLAIS 


DLLFLLTLPF MAHYAANENV 


huCCR5 


LYSLVriFGF VGNMLVILIL 


INCKRLKSMT 


DIYLLNLAIS 


DLFFLLTVPF WAHYAAAQWD 


ratCCR2 


LYSLVFIFGF VGNMLVIIIL 


ISCKKLKSMT 


DIYLFNLAIS 


DLLFLLTLPF WAHYAANEWV 


murCCR2 


LYSLVFIFGF VGNMLVIIIL 


IGCKKLKSMT 


DIYLLNLAIS 


DLLFLLTLPF WAHYAANEWV 


huCCR2b 


LYSLVFIFGF VGNMLWLIL 

X 


INCKKLKCLT 


nTYT.TXT.ATS 


DI.T.FI.TTI.PT. KAHSAANEKV 






II 




121 






180 


ratCCR5 


FGNIMCKLFT GLYHIGYFGG 


IFFIILLTID 


RYLAIVHAVF 


AFKARTVTFG VITSWTWW 


murccr5 


FGNIMCKVTT GLYHIGYFGG 


irFIILLTID 


RYLAIVHAVF 


ALKVRTVNFG VITSWTWAV 


huCCRS 


FGNTMCQLLT GLYFIGFFSG 


IFFIILLTID 


RYLAWHAVF 


ALKARTVTFG WTSVITWW 


ratCCR2 


FGNIMCKLFT GLYHIGYFGG 


IFFIILLTID 


RYLAIVHAVF 


AFKARTVTFG VITSWTWW 


muzCCR2 


FGNIMCKVTT GLYHIGYFGG 


irniLLTID 


RYLAIVHAVF 


ALKARTVTFG VITSWTWW 


huCCR2b 


PRNAMCKI.FT RI.YHTRYFGG 


IFFTTI.I.TTn 


RYLAIVHAVT 


AIJCARTVTFR WTSVTTWt.V 




III 








181 






240 


ratCCR5 


AVFVSLPEII FMRSQKEGSH 


YTCSPHFLHI 


QYRFWKHFQT 


LKMVILSLIL PLLVMVICYS 


murcci5 


AVFASLPEII FTRSQKEGFH 


YTCSPHFPHT 


QYHFWKSFQT 


LKMVILSLIL SLLVMVICYS 


huCCR5 


AVFASLPGII FTRSQKEGLH 


YTCSSHFPYS 


QYQFWKNTQT 


LKIVILGLVL PLLVMVICYS 


raCCCR2 


AVFASLPGII FTKSEQEDDQ 


HTCGPYFP . . 


. .TIWKNFQT 


IMRNILSLIL PLLVMVICYS 


murCCR2 


AVFGSLPGII FTKSKODDHH 


YTCGPYTT. . 


. .QmKNFQT 


IMRNILSLIL PLLVMVICYS 


huCCR2b 


XVFASVPGIT FTKCOKEDSV YVCGPYFP . . 


. .RGHKKTHT 


IMRNILGLVT. PLLIMVICYS 




IV 






V 




241 






300 


ratCCRS 


GILNTLFRCR NEKKRHRAVR 


LirAIMIVYT 


LFHTPYNIVL 


LLTTFQEYFG LNNCSSSNRL 


murccrS 


GILHTLFRCR NEKKRHRAVR 


LIFAIMIVYF 


LFHTPYNIVL 


LLTTFQEFFG LNNCSSSNRL 


huCCR5 


GILKTLLRCR NEKKRHRAVR 


LIFTIMIVYT 


LFNAPYNIVL 


LLNTFQEFFG LNNCSSSNRL 


ratCCR2 


GILHTLFRCR NEKKRHRAVR 


LIFAIMIVYF 


LFHTPYNIVL 


FLTTFQEFLG MSNCWDMHL 


murccr2 


GILHTLFRCR NEKKRHRAVR 


LIFAIMIGYF 


LFHTPYNIVL 


FLTTFQESLG MSNCVXDKHL 


huCCR2b 


GILKTLLRCR NEKKRHRAVR 
301 


VIFTIMIVYF 


T.FWTPYNIVT 


TiTiNTFQEFFG LSNCESTSQL 






VI 


360 


ratCCRS 


DQAMQVTETL GMTHCCLNPV 


IYAFVGEKFR 


NYLSVTFRKH 


IVKRFCKHCS IFQQVNPDRV 


murccrS 


OQAMQATETL GMTHCCLNPV 


IYAFVGEKFR 


SYLSVFFRKH 


MVKRFCKRCS IFQQDNPDRA 


huCCR5 


DQAMQVTETL GMTHCCINPI 


IYAFVGEKFR 


NYLLVFFQKH 


IAKRFCKCCS irQQEAPERA 


ratccr2 


DQAMQVTETL GMTHCCVNPI 


IYAFVGEKFR 


RYLSirFRKH 


IAKNLCKQCP VFYRETADRV 


murCCR2 


DQAMQVTETL GMTHCCINPV 


IYAFVGEKFR 


RYLSIFFRKH 


IAKRLCKQCP VFYRETADRV 


huCCR2b 


DOATOVTETL GMTHCCINPI 


IYAFVGEKrn 


RYLSVFFRKH 


ITKRFCKQCP VFYRETVDGV 




VIZ 








361 377 








ratCCRS 


SSVYTRSTGE QEVSTGL 








murccrS 


SSVYTRSTGE HEVSTGL 








huCCRS 


SSVYTRSTGE QEISVGL 








ratCCR2 


SSTFTPSTGE QEVSVGL 








murCCR2 


SSTFTPSTGE QEVSVGL 








huCCR2b 


TSTNTPSTGE QEVSAGL 









Figure 3-1. Sequence Comparison of Rat and Murine C-C Chemokine Receptors 
Multiple alignment of two rat CC chemokine receptor amino acid sequences (ratCCR2 
and ratCCR5) with the two functional human CC chemokine receptor (huCCR2 and 
huCCR5) and the two murine CC chemokine receptor (murCCR2 and murCCR5). 
Alignment was achieved using the PILEUP algorithm of the GCG package (39). Lines 
denote putative transmembrane regions. Genbank Accession Numbers: 
(huCCR2b:U03905, huCCR5:X91492, murCCR2:U47035, murCCR5:U47036, 
ratCCR2:U77349, ratCCR5:U77350). 






57 



Table 3-1. Amino Acid Identities of the Rat, Murine and Human Chemokine Receptors, 
CCR2 and CCR5 





Murine ortholog 


Human ortholog 


Rat CCR2 


93% 


79% 


Rat CCR5 


92% 


83% 











58 



0.45 —i 

AF 
0.35 —J 




2 min 




2 4 6 8 10 
murMCP-1 (nM) 



Figure 3-2. Calcium Mobilization Response to MCP-1 By HEK293 Cells Expressing Rat 

CCR2. 

HEK293 cells stably expressing rat CCR2 were prepared as outlined in chapter two. 

Transfected cells were loaded with FURA-2A/M. FURA-2 fluorescence was monitored 

before and after addition of MCP-1 . Panel A shows a representative trace. Panel B 

depicts the dose-dependent stimulation of intracellular calcium mobilization by MCP-1 in 

cells expressing rat CCR2. 









59 



murMIP-la 
(500 nM) 

I 



murMIP-ip 
(500 nM) 





Figure 3-3. Functional Responses of Cells Expressing Rat CCR5 

Xenopus laevis oocytes expressing rat CCR5 were prepared as described in chapter two. 

These oocytes were exposed to 500nM murine MP- la (A) or 500 nM murine MP-1 p 

(B). The current induced was monitored on a dual beam storage oscilloscope linked to an 

IBM-PC. 



60 




b *JJfo 



3.2. 
2.9. 

2.7' 



WW 

m —12 

— 7 

— 5 

— 3 



1.1— 



65 °C 



50 °C 



Figure 3-4. Southern Blot Analysis of Restriction Endonuclease-Digested Rat Genomic 
DNA. 

Wistar rat genomic DNA (15 ug) was completely digested with the indicated restriction 
endonucleases and then subjected to Southern blot analysis using the 1.5 kbp [ 32 P]- 
radiolabeled Clal/BamHI fragment of rat CCR5 and the method of Church and Gilbert. 
Final wash temperatures were 65°C (A) and 50°C (B). The blots were exposed to Kodak 
XAR film with an intensifying screen at -80 °C for 1-3 days. The sizes of the hybridizing 
bands are indicated (in kbp) and were estimated by comparison to a GIBCO/BRL 1 -kbp 
ladder. 



61 



# 



* 



£ 









& 
& 
$- 






CCR2 






— CCR2 



L32 







«■! 



jm 



— L32 



Figure 3-5. Rat CCR2 RNA Tissue Distribution in Rat Solid Organs and Cells 
Two ug of total RNA from rat tissues or cells were subjected to RNase protection assay 
(see chapter two). The RNAs were hybridized simultaneously with 32 P-radiolabeled 365- 
base pair rat CCR2 and 93-base pair ribosomal RNA (L32) riboprobes. The dried gel was 
exposed to Kodak X-RP film at -80 °C for 72 hours. 



62 



> / #^ J 






L. 

0. 



J 



CCR5 



GAPDH 




— CCR5 



— GAPDH 



Figure 3-6. Rat CCR5 mRNA Tissue Distribution in Rat Solid Organs and Cells 
Two ug of total RNA from rat tissues or cells were subjected to RNase protection assay 
(see chapter two). The RNAs were hybridized simultaneously with [ 32 P]-radiolabeled 230 
bp rat CCR5 and 1 14 bp GAPDH riboprobes. The dried gel was exposed to X-ray film at 
-80 °C for 10 hours. 



63 



IFN-y 



28 S- 



18 S— 




cyclophilin §•§§ 



Figure 3-7: Dose-Dependent Increase in Rat CCR5/2 mRNA by Interferon-y (EFNy) 
Treated Microglia 

Northern analysis of rat CCR5/2 mRNA in primary cultures of rat microglia treated with 
IFNy. Cultured microglia were incubated for six hours with DMEM (control) or DMEM 
containing the specified concentration of IFNy. At the end of the incubation, total RNA 
was isolated from the culture dishes using the Trizol Reagent (Gibco/BRL). Ten ug of 
total RNA from each group was subject to Northern analysis. Immobilized RNA was 
hybridized with 32 P-labelled CCR5 (Clal/BamHI fragment) by the method of Church and 
Gilbert. Final wash temperature was 65°C. The positions of migration of 18 S and 28 S 
ribosomal RNA are shown. Exposure to Kodak X-AR film was for 3 days (-80°C). The 
lower panel depicts a subsequent hybridization of the blot with 32 P-labelled DNA 
encoding rat cyclophilin and was used to control for lane loading. Exposure time for the 
cyclophilin hybridized membrane was 1 8 hours. 



64 



-w ATA 






■^ ^ o" a" 

ft.GOv V v «• <$• 



XI 
O 



fr> 




£ o°c? n- 'v* *• *• *• 



«* ' 



*£ 



L32-^ 



MM 



# 




CCR2 



CCR5 



N* °J* ^' ©y 



_•. _•. 




p- CXCR4 



RBS11 



cyclophiiin 

Figure 3-8. Expression of CCR2, CCR5, CXCR4, and RBS1 1 mRNAs in Lumbar Spinal 
Cords of EAE Rats as Detected by RNase Protection Assay or Northern Blot Analysis 
EAE was induced in Lewis rats and animals were scored for clinical symptoms as 
described in chapter two. Total RNAs from control and EAE rat spinal cords (5 ug for 
RNase protection assay and 10 ug per lane for Northern blot analysis) were subjected to 
RNase protection assay (upper panel) and Northern blot analysis (lower panels) as 
described in chapter 2. Exposure time for the RNase protection assay gel was 18 hours 
(at -80 °C). Exposure times for the Northern blots were 18 hours (at -80 °C) for the 
RBS1 1 and cyclophiiin hybridized membranes, and 7 days (at -80 °C) for the CXCR4 
hybridized membrane. 



CHAPTER 4 

ROLE FOR NEURONALLY-DERIVED FRACTALKINE IN MEDIATING 

INTERACTIONS BETWEEN NEURONS AND CX3CR1-EXPRESSING 

MICROGLIA— AN IN VIVO STUDY IN THE FACIAL MOTOR NEURON 

REGENERATION PARADIGM 



Introduction 

Among the four subfamilies of chemokine peptides, the newly identified 
chemokine termed fractalkine/neurotactin (Bazan et al., 1997 and Pan et al., 1997) has 
some very unique properties. Its full-length amino acid sequence is comprised of five 
regions: a signal peptide, a chemokine domain, a highly glycosylated mucin-like stalk 
which tethers the chemokine domain with a transmembrane domain and a short 
intracellular region. There are three amino acids in between the first two of the four 
highly conserved cysteine residues in its chemokine domain which makes it the first 
member of the CX3C chemokine subfamily. This peptide can exist as both secreted 
soluble and membrane anchored forms. The soluble form is chemotactic for monocytes 
and lymphocytes while the membrane anchored form can promote a strong adhesion of 
these leukocytes to endothelial cells. Unlike most chemokine peptides, fractalkine is 
expressed in non-hematopoietic tissues including brain, kidney, lung, heart and adrenal 
gland. 

An "orphan" rat G-protein coupled receptor termed RBS 1 1 has a tissue 
distribution pattern that is very similar to fractalkine (Harrison et al., 1994). It is widely 
expressed in the adult rat. Although its amino acid sequence is most similar to other 

65 



66 

chemokine receptors, functional screening assays using a variety of CC and CXC 
chemokine peptides failed to identify its ligand. The similar tissue distribution pattern of 
fractalkine and RBS1 1 prompted us to hypothesize that RBS1 1 is the receptor for rat 
fractalkine. The human ortholog of rat RBS1 1, termed V28, was also identified (Raport 
et al., 1995 and Combadiere et al., 1995). Recently, studies identifying V28 to be a 
receptor for human fractalkine have been reported (Imai et al., 1997, Combadiere et al., 
1998). V28 is designated human CX3CR1. 

In our continuous effort to characterize the role(s) of chemokines and chemokine 
receptors in the CNS, we employed an animal model — the facial motor neuron 
regeneration model — to explore the expression site(s) of the chemokines and chemokine 
receptors in the CNS. A transection of the facial nerve in the periphery can trigger a 
dramatic response in the facial motor nucleus (FMN) of the CNS. This response includes 
microglia activation and proliferation as well as astrocyte activation. Ultimately, the 
damaged motoneurons are regenerated. The advantage of this model is that one can 
challenge the brain tissue without breaking the blood brain barrier. Therefore, only 
endogenous cells are responsible for any reactions in the facial motor nucleus in the CNS. 
The other phenomenon that is associated with this model is so called "synaptic stripping" 
where activated microglia occupy perineuronal positions, forming a sheath around the 
motoneurons and displacing synaptic terminals from the surface of regenerating 
motoneurons (Blinzinger and Kreutzberg., 1968, Graeber and Streit, 1990a, Graeber et 
al., 1990b). 

Microglia activation in FMN after facial nerve transection was determined not 
only by morphological but also immunohistochemical studies. After axotomy, microglia 



67 

antigenic profiles have changed dramatically indicative of an activated state of these cells. 
Increase of CR3 is found as early as 24 hours after axotomy (Graeber et al., 1988a). De 
novo synthesis of MHC (major histocompatibility complex) class I occurrs around 3-4 
days after the lesion followed by the expression of MHC class II and CD4 antigens (Streit 
et al, 1988, Streit et al., 1989,). These findings demonstrate that microglia can express 
molecules involved in communication with other immune responsive cells. However, 
since there is no inflammation and leukocyte infiltration in this paradigm, the relevance 
of these molecules in the regeneration process is not apparent. 

After facial nerve transection, the production of a number of cytokines, 
neurotrophic factors and cell matrix proteins by microglia in the lesioned FMN is 
elevated including interleukin IL-6 (Kiefer et al., 1993), TGF-pM (Kiefer et al., 1993), 
vimentin (Graeber et al., 1988b), and a polymeric ECM protein TSP (thrombospondin, 
Moller et al., 1996). TSP has been shown to promote neuritic outgrowth from neurons in 
the in vitro system (Lawer et al., 1986). The up-regulation of these molecules may play 
an important role for microglia to elicit their function as neuroprotective cells in the 
motoneuron regeneration paradigm. 

Since microglia are immunoresponsive cells in the CNS and chemokine peptides 
and receptors are immunologically relevant molecules, we hypothesized that chemokines 
and/or their receptors are expressed in microglia and play a role in microglia function. In 
this study, we examined the expression of rat fractalkine and its receptor CX3CR1 in the 
FMN after facial nerve transection. 









68 
Results 

Molecular Cloning of Rat Fractalkine 

A rat brain cDNA library (Strategene, CA) was screened by hybridization using a 
rat factalkine cDNA probe. The hybridization probe was generated by PCR using primers 
derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine 
cDNA was sequenced by standard methods and conceptualized protein sequence is shown 
in Figure 4-1. The conceptualized amino acid sequence derived from the cDNA predicts 
a 392 amino acid protein with a molecular mass of 42,161 Da. The amino acid sequence 
identities of rat fractalkine with human and murine forms are 64% and 81%, respectively. 
Analogous to these human and murine proteins, rat fractalkine contains a signal peptide, 
chemokine module, mucin-like stalk, transmembrane spanning region, and a short 
intracellular C-terminus. 
Tissue Distribution of Rat Fractalkine 

In collaboration with Dr. Lili Feng (The Scripps Research Institute), we carried 
out RNase protection assays in order to determine the tissue distribution of rat fractalkine. 
Analogous to its human and murine orthologs, rat fractalkine is expressed in a number of 
non-hematopoietic tissues including kidney, lung, heart, and adrenal gland. Among the 
tissues that we've examined, brain was identified as the most abundant source of rat 
fractalkine (Figure 4-2). 
Distribution of Rat Fractalkine in the Brain 

In situ hybridization analysis revealed that rat fractalkine is highly expressed in 
discrete regions of rat brain including cortex, hippocampus, caudate putamen, thalamus, 



69 

and olfactory bulb (Figure 4-3) and was significantly lower or absent in cerebellum and 
white matter regions including corpus collosum and fimbria/fornix. 
Identification of RBS1 1 As the Receptor for Rat Fractalkine 

Figure 4-4 demonstrates the ability of radiolabeled chemokine domain of 
fractalkine bind to RBS 1 1-transfected CHO cells. Wild type CHO cells did not display 
any significant binding. Inhibition binding constants (IC50s) of 2 and 3 nM for the 
chemokine domains of rat and human fractalkine, respectively, were determined. Other 
CC or CXC chemokine peptides that we tested, including MlP-la, RANTES, MCP-1 
MTP-2, and SDF-1, did not displace the binding of labeled fractalkine to RBS1 1 
expressing CHO cells. This evidence further corroborated that the binding between 
fractalkine and RBS1 1 transfected cells was specific. Figure 4-5 depicts the functional 
properties of RBS 1 1 in the transfected CHO cells. Application of either soluble or 
membrane-tethered forms of fractalkine stimulated a transient elevation of intracellular 
calcium levels in the RBS1 1 expressing CHO cells. These data, collected by Dr. Jeffrey 
Harrison, unequivocally identified that RBS1 1 is a functional receptor for rat fractalkine. 
Expression and Regulation of Rat CX3CR1 mRNA in Cultured Microglia 

To identify the expression site(s) of rat CX3CR1, Northern blot analysis was 
carried out. Primary cultures of rat microglia were incubated for eight hours with DMEM 
(control) or DMEM containing increasing concentrations of TGF-(3. At the end of the 
incubation, total RNA was isolated from these microglia and subjected to Northern blot 
analysis. Figure 4-6 depicts constitutive expression of rat CX3CR1 mRNA in cultured 
microglia. Steady state levels of CX3CR1 mRNA present in the cultured microglia were 
dose-dependently increased by TGF-P treatment. 



70 

Expression and Regulation of Rat CX3CR1 in vivo in the Facial Motor Nucleus After 
Facial Nerve Transection 

The facial motor neuron regeneration model was used to determine the 
localization and regulation of expression of CX3CR1 in rat CNS. In situ hybridization 
analysis was used to assess the expression of CX3CR1 in the facial motor nucleus over a 
2 1 day period during which the facial motor neurons undergo regeneration. At time 
points of 4, 7, and 14 days post-axotomy, the levels of CX3CR1 hybridizing mRNA were 
significantly elevated in the injured facial motor nucleus (Figure 4-7), as compared to the 
contralateral nucleus. Highest levels of CX3CR1 mRNA were days 4 and 7 post- 
axotomy. At the 21 day time point CX3CR1 mRNA had returned to a level comparable 
to that detected in the contralateral nucleus. The graph depicts quantitative analysis of rat 
CX3CR1 mRNA hybridizing signal in the FMN of control and lesioned side. 

The widespread distribution of rat CX3CR1 in normal rat brain (Harrison et al., 
1 994) and its presence on cultured microglia indicate that the CX3CR1 gene might also 
be expressed on these cells in vivo. To determine the expression site(s) of rat CX3CR1 in 
the CNS, in situ hybridization analysis was combined with histochemical staining using 
sections of brain stem containing FMN four days after facial nerve transection. Lectin 
(from Griffonia simplicifolia, GSA-I-B4) staining was used to identify microglia cells 
whereas anti-GFAP (glial fibrillary acidic protein) antibody was applied to identify 
astrocytes. Figure 4-8 demonstrates the colocalization of rat CX3CR1 mRNA 
hybridizing signals (silver grains) with lectin staining positive microglia but not anti- 
GFAP positive astrocytes (data not shown) in the FMN before and after axotomy. This 
data confirmed that rat CX3CR1 is expressed principally by microglia in vivo. 



71 • 

Quantitative analysis indicated that, in the control FMN, only 30% of the lectin staining 
microglia were CX3CR1 positive (a cluster of>10 silver grains over a single cell) whereas 
in the lesioned FMN, there is approximately 60% of the lectin staining microglia 
expressing CX3CR1. In addition, the intensity of CX3CR1 mRNA signal, represented by 
numbers of silver grains found over the microglia, appear to be comprable between the 
control and lesioned FMN. This suggests that CX3CR1 mRNA expressed by each single 
microglia did not change after facial nerve axotomy. 
Expression of Rat Fractalkine in FMN After Facial Nerve Transection 

The expression of fractalkine mRNA in the facial motor nucleus following 
axotomy was also examined. In situ hybridization analysis demonstrated that the 
motoneurons were the primary source of fractalkine mRNA (Figure 4-9 A-D). 
Fractalkine positive cells were not colocalized with either lectin positive microglia or 
anti-GFAP positive astrocytes. A decrease in the hybridization signal in motoneurons 
four days after axotomy was also evident. Western blot analysis done by Elizabeth Wees 
in our laboratory demonstrated that lower molecular mass forms of rat fractalkine protein 
levels were upregulated in the lesioned FMN. 

Discussion 

The expression of some of the chemokine and chemokine receptors in the CNS 
has been reported by a number of different groups. In this study, we isolated the rat 
ortholog of human and murine fractalkine by homology screening technique and 
examined the expression and regulation of rat fractalkine and its receptor rat CX3CR1, in 
the facial motor neuron regeneration paradigm. Consistent with its human and murine 



72 

orthologs, rat fractalkine gene encodes a protein that has a chemokine domain followed 
by a highly glycosylated mucin-like stalk, a transmembrane domain and a short 
intracellular domain. Its mRNA can be found in some non-hematopoietic tissues 
including lung, heart, kidney, and adrenal gland. However, its expression is most 
abundant in the brain where it is expressed principally by neurons. 

By in situ hybridization analysis, we demonstrated that the expression of CX3CR1 
mRNA was transiently up regulated in the facial motor nucleus (FMN) after axotomy. 
The time course of this up-regulation parallels the transient increase in microglial cell 
numbers after axotomy. It is also known that, at this time point, the majority of 
axotomized motoneurons are tightly ensheathed by the perineuronal microglial cells 
(Kreutzberg 1996, Streit 1996). In an in vivo study, Kiefer et al. (1993) demonstrated 
that TGF-P mRNA level was up regulated transiently in the lesioned FMN. It peaked 
between days 2-7 and gradually comes down after that and returned to normal by three 
weeks post-lesion. Our in vitro study indicated that cultured microglia constitutively 
express rat CX3CR1 . The expression is up regulated in a dose dependent manner after 
TGF-P treatment. Collectively, it can be speculated that TGF-P may play an important 
role in regulation of microglial expression of CX3CR1 in vivo as well. 

Compared to the other CNS inflammatory models such as EAE, the motor neuron 
regeneration model provides the advantage of confined cell population within the CNS 
because of the intact blood brain barrier. This brings a great advantage in our effort to 
identify the cellular localization of the genes that we are interested in. Our data 
unequivocally demonstrated that in the FMN, microglia are the principal source of 
CX3CR1 whereas fractalkine is primarily expressed by motor neurons. Our collaborator 



73 

Dr. Lili Feng's group collected similar data demonstrating that neurons are the source of 
fractalkine in cortex, hippocampus and thalamus of adult rat (data not shown). The 
interaction between fractalkine and its receptor CX3CR1 may be an important mechanism 
by which neurons communicate with microglia and elicit their functional roles during 
facial motor neuron regeneration. 

The present study did not define the specific form(s) of rat fractalkine that was 
expressed in FMN. Our functional analysis shown in figure 4-2 indicated that the 
response of CX3CR1 expressing cells to fractalkine various based upon the different 
forms of the peptide (membrane vs. soluble). Membrane-anchored forms of fractalkine 
activated a greater calcium mobilizing response compare to soluble forms. Previous 
studies (Imai et al., 1997) demonstrated that a soluble form of fractalkine stimulates 
chemotaxis while a membrane anchored form promotes adhesion. Thus, it is conceivable 
that the larger calcium response triggered by membrane anchored form is related to cell 
adhesion whereas the minor response stimulated by soluble form results in chemotaxis. 
Alternatively, these forms of fractalkine may serve other functions for the CX3CR1 
expressing cells than chemotaxis or adhesion. Analogous to the receptor-transfected 
CHO cell model, studies carried out by our collaborator Dr. Kevin Bacon's group 
demonstrated that differential stimulation of microglia intracellular calcium mobilization 
by fractalkine was dependent on the form present (data not shown). Membrane-anchored 
forms of fractalkine stimulated robust changes in intracellular calcium levels while a 
soluble form of fractalkine had no effect. Our speculation based upon these data is that 
potentially, fractalkine-expressing neurons may function in regulating CX3CR1 
expressing microglial activity by providing different forms of fiactalkine to the 



74 

microenvironment. However, the physiological relevance of these relative intracellular 
calcium levels in both transfected cells and microglia still remains to be determined. We 
hypothesize that the soluble form of fractalkine stimulates an unknown signaling pathway 
which results in microglial chemotaxis towards facial motor neurons whereas the 
membrane anchored form elevates intracellular calcium levels which functions to 
promote the adhesion of microglia to these neurons and causes the ensheathement of 
neurons by microglia, known as "synaptic stripping". 

In contrast to the up regulation of CX3CR1 mRNA in FMN after nerve 
transection, the mRNA level of fractalkine is down-regulated four days after the lesion. 
However, our Western blot analysis indicates the up-regulation of some lower molecular 
mass forms of fractalkine protein in the lesioned FMN (data not shown). This 
discrepancy between mRNA and protein levels of rat fractalkine in FMN after nerve 
transection is suggestive of the dynamic regulation of fractalkine expression in the CNS. 

The constitutive expression of fractalkine and CX3CR1 in discrete regions of the 
CNS indicates that they are involved in fundamental processes of communication 
between neurons and microglia under both pathological as well as physiological 
circumstances. A better understanding of the functional roles of these molecules will 
eventually lead to the development of therapeutic reagents that can interrupt disease 
processes that these molecules are involved in. 



75 



Rat Fractalkine (CX3C) 



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Figure 4-1. Amino Acid Sequence of Rat Fractalkine (Genbank accession number 
R75309) 

A rat brain cDNA library (Strategene, CA) was screened by hybridization using a rat 
factalkine cDNA probe. The hybridization probe was generated by PCR using primers 
derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine 
cDNA was sequenced by standard methods (Sanger dideoxy chain termination, 
Sequenase kit). 



76 



Frac 



L32 




-Frac 



-L32 



Figure 4-2. RNase Protection Analysis of Fractalkine mRNA in Various Tissues of Adult 

Rat 

Five ug of total RNA from rat tissues or cells were subjected to RNase protection assay 

(see Materials and Methods). The RNAs were hybridized simultaneously with 32 P- 

radiolabeled 400-base pair Ncol fragment of rat fractalkine cDNA and 93-base pair 

ribosomal RNA (L32) riboprobes. The dried gel was exposed to Kodak X-RP film at -80 

°C for 72 hours. 



77 



MBKM 


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mi 










MM 

mf<£e ■ •• •-,."*■• • 


fejf ca3 • "•:' •* ' 




cbm 


? it 



Figure 4-3. Distribution of Rat Fractalkine mRNA in Adult Rat Brain 
Hybridization analysis using "anti-sense" (A) and "sense" (B) [ 35 S]-UTP-labeled 
riboprobes generated from rat fractalkine cDNA. Hybridized horizontal sections were 
exposed to film for 18 hours (abbreviations used: Olf: olfactory bulb; ctx: cortex; cc: 
corpus collosum; CPu: caudate putamen; Th: thalamus; ca3: ca3 region of the 
hippocampus; gc: granule cell layer of the hippocampus; CG: central gray; cbm: 
cerebellum^ 



78 






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80 

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ffl 

.o 40H 

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02 7 



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[Inhibitor], log M 



Figure 4-4. Whole Cell Radioligand Binding Analysis of CHO-CX3CR1 -Expressing 
Cells v 6 

Competition of 1 nM labeled peptide with the chemokine domain of rat fractalkine (a), the 
chemokine domain of human fractalkine $, rat MCP-1 (0, rat MP- la ($, rat RANTES (• 
), rat SDF-1 (▼), and murine MIP-2 (a). Wild type CHO cells did not display any 
significant specific binding of 125 I-fractalkine (7). Data were calculated as the fraction of 
specific bmdmg in the presence of the specified concentration of unlabeled chemokine 
peptides^ [CPMs bound per 3 million cells were: Total: 7945 + 428; Non-specific: 2078 + 
231, N - 7]. The results (from triplicate determinations) are presented as the mean + 
S.E.M. of 3 experiments (rat fractalkine), 4 experiments (human fractalkine) or the mean 
of two experiments (all other chemokines). The calculated IC50s for inhibition of labeled 
peptide bmdmg by the chemokine domains of either rat or human fractalkine are 2 2 nM 
(95% confidence interval: 0.9 - 5.3 nM) and 2.9 nM (95% confidence interval: 1.9 - 4 5 
nM), respectively. 



79 






\** 




Iv 



vll . 




III 

0.2. 

1 mln. 
W 




[Fractalkine], nM 

Figure 4-5. Calcium Mobilization Response to Different Forms of Fractalkine by CHO 
Cells Expressing Rat CX3CR1 

Representative traces of CHO-CX3CR1 expressing cells in response to application of 
either 293T cells transiently expressing rat (i, ii) or human fractalkine (iv, v), or the 
chemokine domain of rat (iii) or human (vi) fractalkine. Wild-type fractalkines (i, iv) or 
mutant fractalkines (ii, v) in which arginine-arginine residues adjacent to the TM domain 
were changed to serine-alanine, were expressed transiently in 293T cells. Lower traces 
depict application of mock transfected 293T cells (vii) to CHO-CX3CR1 -expressing cells 
or addition of the chemokine domain of rat fractalkine (viii), or the wild-type membrane- 
tethered form of rat fractalkine (ix) to wild-type CHO cells. The graph depicts 
concentration-dependent effect of the chemokine domain of human fractalkine on 
intracellular calcium levels in CHO-CX3CR1 -expressing cells. Data are plotted as the 
fraction of the maximal response seen in the presence of a high dose of fractalkine. 
Values are the mean of at least two determinations at each concentration. The calculated 
EC50 from these experiments is 3 nM. 



8(1 




-28S 



-18S 



cyclophilin 



Figure 4-6. Dose-Dependent Increases in Rat CX3CR1 mRNA by Transforming Growth 
Factor-p (TGF-P) Treated Microglia 

Northern analysis of rat CX3CR1 mRNA in primary cultures of rat microglia treated with 
TGF-p. Cultured microglia were incubated for eight hours with DMEM (control) or 
DMEM containing the specified concentration of TGF-p. At the end of the incubation, 
total RNA was isolated from the culture dishes using the Trizol Reagent (Gibco/BRL).' 
Ten ug of total RNA from each group was subject to Northern analysis. Immobilized 
RNA was hybridized with 32 P-labelled 1.1 kbp CX3CR1 cDNA fragment by the method 
of Church and Gilbert. Final wash temperature was 65°C. The positions of migration of 
1 8 S^and 28 S ribosomal RNA are shown. Exposure to Kodak X-AR film was for 4 days 
(-80°C). The lower panel depicts a subsequent hybridization of the blot with 32 P-labelled 
DNA encoding rat cyclophilin and was used to control for lane loading. Exposure time 
for the cyclophilin hybridized membrane was 18 hours. 



81 





5 10 15 20 25 

Days Post-Lesion 




Figure 4-7. In Situ Hybridization Analysis of CX3CR1 Expression in the Rat Facial 
Nucleus After Motor Neuron Axotomy 

In situ hybridization was performed on rat brainstem sections from animals sacrificed at 1 
(A), 4 (B), 7 (C), 14 (D), and 21 (E) days post nerve transection using [ 35 S]-UTP-labeled 
riboprobe. Panel F depicts a quantitative analysis of the hybridization signal on the 
operated (•) and contralateral (o) sides. Results are presented as the mean + S.E.M. of 
data derived from at least three sections from three different animals. The hybridization 
signal was specific for CX3CR1 as no increase in signal was observed using a "sense" 
riboprobe (H) when compared to a corresponding "anti-sense" riboprobe hybridized- 
section (G). 



82 



# 



/ N 

A 

H 



#-* 









B 









^> 




Figure 4-8. Colocalization of Rat CX3CR1 mRNA With Microglial Cells 
Rat CX3CR1 mRNA hybridization signals (silver grains) from "anti-sense" [ 33 P]-UTP- 
labeled riboprobe hybridized-sections are found principally over lectin (GSA-I-B4) 
staining microglia in the neuropil of the unoperated facial motor nucleus (B). Several 
CX3CR1 mRNA containing microglial cells are found perineuronally [arrows in (A)] 
surrounding axotomized motorneurons (N: motor neuron cell body). (C) "Sense" [ 33 P]- 
UTP-labeled riboprobe-hybridized section showing only a few scattered silver grains. 






83 





H 






* 



■JP./* 






I V 




Figure 4-9: Fractalkine Expression in the Rat Facial Nucleus After Motor Neuron 

Axotomy 

In situ hybridization was performed on rat brainstem sections from animals sacrificed 4 

days post nerve transection. Hybridization analysis of the contralateral (A and B) and 

ipsilateral (C and D) facial motor nuclei using "anti-sense" (A and C) and "sense" (B and 

D) [ P]-UTP-labeled riboprobes generated from rat fractalkine cDNA. Hybridization 

signals are found principally over the motor neurons. 



CHAPTER 5 
DIFFERENTIAL EXPRESSION OF CX3CR1 IN TWO RAT GLIOMA MODELS- 
ROLE FOR TGF-p AND CX3CR1 IN MICROGLIAL CELL DEATH WITHIN RAT 

BRAIN TUMORS 



Introduction 

It has been known for sometime that tumor growth in vivo can create an 
immunosuppressive environment (Sawamura and De Tribolet, 1 990). What role 
chemokines and their receptors play in this environment is a question that needs to be 
addressed. 

Some tumor cell lines and tumors have been found to express chemokine 
peptides. For instance, IL-8 was found to be expressed in lung carcinoma, osteosarcoma, 
melanoma, renal carcinoma, fibrosarcoma, astrocytoma and glioblastoma cell lines; Gro 
peptides were found in melanoma, renal carcinoma and bladder carcinoma cell lines; 
MCP-1 expression was seen in fibrosarcoma, glioma, histocytoma, melanoma, 
osteosarcoma and sarcoma cell lines. In vivo studies revealed that MCP-1 is expressed in 
ovarian cancer (Negus et al., 1995), prostate adenocarcinoma (Mazzucchelli et al., 1996), 
and human invasive ductal breast cancer (Valkovic et al., 1998). In these studies, ovarian 
cancer cells and breast cancer cells were found to be the sources of MCP-1 whereas 
prostate adenocarcinoma cells were not. The expression and function of CXC 
chemokines in tumor development have been studied rather extensively mainly based on 
their angiogenic or antiangiogenic functions. IL-8 and IP 10 have been found in human 



84 



85 

non-small cell lung cancer tissues. Immunohistochemical studies revealed that neoplastic 
cells are the principal sources of these peptides (Yatsunami et al., 1998, and Arenberg et 
al., 1996). Multiple studies carried out by a number of groups demonstrated that IL-8 
promotes tumorigenesis because of its angiogenic function whereas IP 10 has tumor 
growth inhibitory function based primarily on its angiostatic activity (reviewed by 
Arenberg et al., 1997). There is evidence to support the speculation that the ELR motif in 
the N-terminus of some of the CXC chemokine peptides plays an important role in the 
angiogenic activity of these peptides and a lack of a ELR motif in other CXC chemokine 
peptides confers their angiostatic activity. The mechanism of these phenomena remains 
to be elucidated. 

There is very limited information regarding the expression of chemokines and 
their receptors in brain tumor tissue. Studies from several different groups have shown 
that several astrocytoma derived cell lines express MCP-1 either constitutively or after 
induction with EL-1 beta and TNF alpha (Kasahara et al., 1991). MCP-1 mRNA and 
protein expression has also been demonstrated in vivo from human glioma tumor cells, 
endothelial cells and possibly infiltrating macrophages (Takeshima et al., 1994). The 
evidence that MCP-1 expression correlated with the degree of macrophage infiltration 
into the tumor (Takeshima et al., 1994) and that MCP-1 containing cyst fluids derived 
from brain tumors exhibited chemotactic activity for macrophages in vitro (Desbaillets et 
al., 1994) suggests that MCP-1 is biologically active in the tumors in vivo. Another study 
carried out by Barnes et al. (1996) demonstrated that astrocytoma cells produced 
RANTES when stimulated with TNF-a or IL-lp. 



86 

The experimental rat glioma models are useful in studying chemokine and 
chemokine receptor expression and function in brain tumors. RG2 and C6 gliomas are 
two very widely used rat models in experimental neuro-oncology (Barth et al., 1998). 
Both gliomas are chemically induced with different immunogenic responses. RG2 
glioma appears to be either weakly or non-immunogenic. This tumor is refractory to a 
variety of therapeutic modalities and has an invasive growth pattern as well as uniform 
lethality. Compared to RG2 glioma, C6 glioma has a potential to evoke an alloimmune 
response partly because it is not syngeneic to any inbred strain. Morioka et al. (1992) has 
reported that in RG2 gliomas, increased numbers of microglia were formed around and 
infiltrated into the tumor. This indicated that tumor growth in the brain causes microglial 
activation and proliferation. However, the question of whether or not these microglial 
cells are functional as tumor surveillance cells remains unanswered. The microglial 
response in C6 glioma has not been carefully studied. 

Our data from the previous chapter demonstrated that CX3CR1 mRNA is 
expressed in microglia in vivo. We then set out to determine if this is also true in tumor 
tissues. The specific aim of this study was to determine the expression and to specify the 
cellular localization of fractalkine and CX3CR1 in experimental rat glioma tissues (RG2 
and C6 gliomas). Results generated by this study may provide more information in 
understanding brain tumor biology as well as some aspects of the function of fractalkine 
and CX3CR1 in the CNS. 



87 
Results 

Expression of Rat CX3CR1 in C6 Glioma 

To examine the expression of rat fractalkine as well as CX3CR1 in RG2 and C6 
gliomas, Northern blot and in situ hybridization analysis were carried out. After tumor 
cell inoculation, female Fisher rats were sacrificed at the point in time when the first C6 
or RG2 inoculated animal developed the first signs of neurological deficit. It normally 
takes 2-3 weeks for RG2 glioma and 4-6 weeks for C6 glioma inoculated rats to develop 
some symptoms. For Northern blot analysis, tumor tissues were dissected out on an ice 
cold surface and surrounding normal tissues were removed. RNA isolation was carried 
out using Trizol reagent. For in situ hybridization, rats were sacrificed as described in 
chapter two and frozen sections were made subsequently. Both Northern blot and in situ 
hybridization analysis demonstrated that these gliomas express little if any fractalkine 
mRNA. C6 tumors, but not RG2 tumors, expresses CX3CR1 mRNA (Figure 5-1, Figure 
5-2). In collaboration with Sharon Walters and Dr. Wolfgang Streit, we also examined 
the ability of these gliomas to express TGF-(3 mRNA. Figure 5-2 (E-F, K.-L) demonstrats 
that RG2 tumors express much higher levels of TGF-(3 mRNA than C6 tumors do. 
Emulsion dipped slides confirmed that tumor cells rather than microglia were the sources 
of TGF-P mRNA (data not shown). 
Rat CX3CR1 mRNA Expression by Microglia in C6 Gliomas 

To identify the cellular localization of CX3CR1 in C6 gliomas, lectin (GSA I-B4) 
staining was carried out in combination with in situ hybridization as described in chapter 
two. Slides were then exposed to Kodak NTB2 autoradiographic emulsion at 4°C for 



88 

approximately 3-4 weeks before being developed. Our data indicated that rat CX3CR1 
mRNA is expressed in microglia within C6 gliomas as clustered silver grains were 
evident over lectin binding positive cells (Figure5-3A). In RG2 gliomas rat CX3CR1 
mRNA is expressed by microglia on the border of the tumor but not lectin positive cells 
within the tumor (data not shown). 
Different Microglial-Infiltrating Patterns Between C6 and RG2 Gliomas 

Lectin (GSA I-B4) staining of both C6 and RG2 gliomas were carried out to 
characterize microglial- infiltrating patterns of these two gliomas. Figure 5-4 
demonstrates that within RG2 tumors, lectin positive microglia tend to cluster together to 
form isolated islands (A); many cells have pyknotic nuclei — one of the characteristics of 
apoptotic cells (B); many areas within RG2 tumors have none or very little microglial 
infiltration (data not shown). On the other hand, inside C6 gliomas, infiltrating microglia 
are evenly distributed throughout the tumor (C); these cells conserve the morphology of 
normal microglial cells (D). 
Microglial Apoptosis Within C6 and RG2 Gliomas 

We further examined apoptosis within C6 and RG2 gliomas by TUNEL staining 
(Terminal Transferase dUTP Nick End-Labelling) as described in chapter two. TUNEL 
is a staining assay that labels cells with fragmented DNA ends, characteristic of apoptotic 
cells. Figure 5-5 demonstrates that within RG2 tumors, TUNEL staining positive cells 
are clustered to form isolated islands similar to what we see in RG2 tumors with lectin 
staining (A). However, in the C6 gliomas that I tested, there are only a few TUNEL 
positive cells (data not shown) and the major areas of the tumor mass were TUNEL 
negative (B). 



89 
Discussion 

Towards our goal for determining expression of fractalkine and CX3CR1 in the 
rat glioma models, my data demonstrated that neither C6 nor RG2 gliomas express rat 
fractalkine mRNA in vivo. While C6 gliomas express relatively high levels of rat 
CX3CR1 mRNA, there is no detectable expression of rat CX3CR1 in RG2 gliomas by 
both Northern blot and in situ hybridization analysis. Infiltrating microglia are the 
primary sources of rat CX3CR1 mRNA in C6 gliomas. 

In another study collaborating with Sharon Walters from Dr. Wolfgang J. Streit's 
laboratory, we found that RG2 gliomas, as compared to C6 gliomas, express very high 
levels of TGF-P mRNA. However, our in vitro study shown in chapter four (Figure 4-6) 
demonstrated that the expression of rat CX3CR1 mRNA in cultured microglia is up- 
regulated in a dose dependent manner by TGF-P treatment. The paradox became 
apparent: since microglia are the primary sources of CX3CR1 and RG2 gliomas express 
very high levels of TGF-p, one would predict that RG2 gliomas are abundant sources of 
rat CX3CR1 mRNA. Hoever, our data indicate the opposite. Besides the argument that 
in vitro systems don't necessarily always represent what happens in vivo, we also looked 
into the alternative explanations. Combination of lectin (GSA I-B4) staining with 
TUNEL staining using RG2 as well as G6 glioma tissues allows us to examine the 
survival state of the infiltrating microglia in these two gliomas. In RG2 gliomas, lectin 
staining and TUNEL staining positive cells form a very similar pattern — clustered 
together to form isolated islands, many of which have comparable shapes; cell 
morphology of lectin positive cells resembles TUNEL positive cells with many pyknotic 



90 

nuclei. These data support my hypothesis that in RG2 tumors, most infiltrating microglia 
are going through apoptosis. On the other hand, in C6 gliomas, microglial cells are 
evenly distributed through out the tumors that have very little apoptotic cells. Taking 
together, these evidences suggest that infiltrating microglia are stimulated to go through 
apoptosis in RG2 gliomas; these apoptotic cells no longer capable of expressing 
CX3CR1. However, infiltrating microglia in C6 gliomas do not go through apoptosis and 
thus, are capable of expressing CX3CR1. 

Based on the results of our in situ hybridization analysis that TGF-(5 mRNA 
expression in RG2 tumors is substantially higher than C6 tumors, we hypothesize that 
TGF-P, may play a very important role in mediating microglial apoptosis in RG2 
gliomas. However, previous studies have given us controversial results. Data from Xiao 
et al. (1997) demonstrated that TGF-P was able to induce cultured microglial apoptosis as 
compared to the control group. A similar study performed by Jochen Gehrmann (1995) 
indicated that TGF-P did not have such an effect on cultured microglia. Some 
preliminary results from my collaborator Sharon Walters did not show any significant 
increase of microglial cell death after TGF-p treatment. Further studies remain to be 
done to clarify this issue. On the other hand, since CX3CR1 expression of cultured 
microglia is elevated as a result of TGF-P treatment, fractalkine/CX3CRl could 
potentially also play a role in mediating microglial apoptosis. 

It is commonly known that tumor growth within a body stimulates inflammatory 
responses. T-lymphocyte infiltration has been found in malignant gliomas (Weller et al., 
1995). Neutrophil infiltration is predicted based on the evidence revealing the expression 
of neutrophil chemoattractant including IL-8 and CINC (Yamanaka et al., 1994, 



91 

Desbaillets et al., 1997, Uehara et al., 1998) in glioma cell lines or glioma tissues. 
Polynuclear cells that have the morphology constant with neutrophils were found in my 
data generated by lectin or TUNEL stainin (Figure 5-4b and 5-5b); this observation leads 
to an alternative explanation of the result. It is possible that TUNEL positive islands in 
RG2 tumors are necrotic foci in fast growing RG2 tumors as a result of inflammation and 
oxygen deprivation within tumor tissue. High levels of H2O2 inside neutrophils 
responded to HRP-congugated lectin and presented false lectin positive signals. To test 
this possibility, double staining of these glioma section with neutrophil specific marker 
and TUNEL labeling could help to identify infiltrating cells and/or apoptotic cells. 

Taken together, this is the first study demonstrating differential expression of 
fractalkine receptor, CX3CR1, in rat glioma models. The different CX3CR1 expression 
may represent different functional roles or different survival states of microglia in 
different rat glioma tissues. Future studies based on these observations could provide 
insights into glioma biology as well as functions of fractalkine and CX3CR1 in tumor 
development. 



92 












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Figure 5-1. Expression of Rat CX3CR1 in C6 Glioma— Northern Blot Analysis 
Northern blot analysis was carried out to examine the expression of rat fractalkine and 
CX3CR1 in cultured RG2 and C6 cells as well as RG2 and C6 gliomas. Tumor tissues 
were dissected out on an ice cold surface and normal tissues were removed. RNA 
isolation was carried out using Trizol reagent. Ten ul of total RNA were loaded in each 
lane. The hybridizations were carried out using the Church and Gilbert (1984) method. 
The blots were hybridized with both 400bp and 750bp fractalkine cDNA fragment (A) or 
a 1 .2kbp rat CX3CR1 cDNA fragment (B). Hybridizing blot was exposed to the film for 
four days at -80°C. The lower panel depicts a subsequent hybridization of the blot with 
J "P-labelled DNA encoding rat cyclophilin and was used to control for lane loading. 
Exposure time for the cyclophilin hybridized membrane was 1 8 hours 



93 















Figure 5-2. Expression of Rat CX3CR1 in C6 Glioma— In Situ Hybridization Analysis 
In situ hybridization analysis was carried out as described in chapter two. RG2 (A-F).or 
C6 (G-L) tumors are probed with antisense (A, C, E, G, I, K) or sense (B, D, F, H, J, L) 
riboprobes of CX3CR1 (A. B, G, H), fractalkine (C, D, I, J) or TGF-p (E, F, K. L) 



94 




Figure 5-3. Expression of Rat CX3CR1 in Infiltrating Microglia in C6 Gliomas 
After exposure to emulsion film for 3-4 weeks, sections containing RG2 or C6 gliomas 
were developed and counter stained with cresyl violet. C6 glioma sections were probed 
with antisense (A) or sense (B) CX3CR1 riboprobe. 







■ 



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Figure 5-4. Lectin (GSA I-B4) Staining of RG2 (A, B) and C6 (C, D) Sections 
Lectin staining, to identify infiltrating microglia in RG2 or C6 gliomas, was carried out as 
described in chapter two. Pictures were taken at different (A, C, lOOx, B, D: 4000x) 
magnifications. Figure 5-4A illustrates one of many fields where lectin positive cells 
clustered to form isolated islands. 



96 










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Figure 5-5. TUNEL (Terminal Transferase dUTP Nick End-Labeling) Analysis in RG2 
and C6 Gliomas 

TUNEL analysis was carried out on sections containing RG2 (A) or C6 (B) gliomas as 
described in chapter two. Panel A is a representative field where TUNEL positive cells 
clustered to form isolated islands in RG2 tumors. Panel B illustrates that very little 
TUNEL positive cells can be seen in C6 tumors. 



CHAPTER 6 

CONCLUSIONS AND FUTURE 

DIRECTIONS 

Since the discovery of chemokine receptors functioning as HTV entry cofactors, 
there has been growing interest in studies of the expression and function of chemokine 
peptides and receptors. Chemokine peptides are primarily found to be involved in 
leukocyte recruitment and activation during inflammatory processes. Recently, these 
molecules have been shown to participate in such diverse processes as development, 
hematopoiesis, angiogenesis, lymphocyte homing and trafficking, and apoptosis. 

Studies carried out in this dissertation were aimed at characterizing the expression 
of chemokine receptors in the central nervous system (CNS) with the goal to begin to 
understand the functional role(s) these molecules perform during neurophatho logical as 
well as neurophysiological conditions. 

In the first part of this study, we isolated two closely related rat CC chemokine 
receptor genes, CCR2 and CCR5, using sequence homology screening techniques. The 
identities of these two receptors were determined based on their high amino acid 
sequence identity to the human orthologs and the functional responses of receptor 
expressing cells to their specific ligands. While both CCR2 and CCR5 are expressed in a 
number of tissues in the periphery, CCR5 is expressed constitutively in the brain and 
cultured microglia. The steady state expression of CCR5 is up regulated after the 
treatment of the cultured microglial cells with IFN-y. An animal model, Experimental 



97 



98 

Allergic Encephalomyelitis (EAE), was utilized to examine the expression of these 
chemokine receptors during inflammation of the CNS. My results revealed elevation of 
the expression of CCR5 and CCR2 mRNA levels in lumbar spinal cords of diseased rats. 
These informations could be useful in developing reagents against MS by interrupting 
signaling of these chemokine receptors. 

To determine expression site(s) of chemokine receptors in the CNS, a non- 
inflammatory CNS injury model — facial motoneuron regeneration model was adopted. 
The advantage of this model is that one can stimulate a CNS reaction with out breaking 
the blood brain barrier. Thus, only endogenous cells are responsible for any changes 
during the reaction. Using this model, I determined expression sites of a newly identified 
chemokine/receptor pair, fractalkine and CX3CR1 . In situ hybridization analysis 
revealed that while neurons express fractalkine, microglia are the principal sources of 
CX3CR1 mRNA. Thus, neurons could activate a microglial reaction by providing certain 
forms of fractalkine whereas microglia may regulate their expression levels of CX3CR1 
to elicit their neuroprotective or neurotoxic functions in responce to changes of the 
microenvironment. These results suggest a potential mechanism by which neurons and 
microglia communicate with each other and play their functional roles under normal or 
diseased situations. 

Since chemokine peptides and receptors are immune relevant molecules and 
development of neoplasms generally involve a host immune responses, I further 
examined the expression of fractalkine and CX3CR1 in two rat glioma models, RG2 and 
C6 gliomas with the attempt to begin to understand the functional roles of these 
molecules in glioma development. Fractalkine was not detectable in either RG2 or C6 






99 



gliomas whereas CX3CR1 mRNA was found in C6 but not RG2 glioma. In situ 
hybridization analysis indicated that CX3CR1 is expressed in microglia. The differential 
expression of CX3CR1 in these gliomas is suggestive of a role for CX3CR1 in regulating 
microglial function in responding to tumor growthin the CNS. 

Future directions based on studies in this dissertation could be aimed at studying 
signal transduction pathways that these chemokine receptors use to elicit their functional 
roles; analyzing the regulation of expression of fractalkine and CX3CR1 mRNA as well 
as protein in vitro and in vivo; examining the regulation of expression and functional 
roles of different forms of fractalkine peptides in normal as well as disease processes; 
investigating the effect of these molecules on regulating the expression of other 
intracellular components. 

To test whether TGF-p\ fractalkine and CX3CR1 are involved in microglia 
apoptosis in these glioma models, we can treat cultured microglia with TGF-(3 plus or 
minus fractalkine and look for the different percentage of apoptotic cells in each group of 
treatment via TUNEL labeling. More convincing evidence has to come from in vivo 
systems. Studies in fractalkine or CX3CR1 "knock out" animals will be very useful in 
testing potential roles of these molecules in regulating microglia function. 

In conclusion, among rat chemokine receptors and peptides that I examined, rat 
CCR5, CX3CR1 and fractalkine are constitutively expressed in the CNS. Expression 
levels were found to be regulated under pathological circumstances. The expression and 
regulation of these molecules in the CNS indicates important functional roles in 
neuropathology as well as neurophysiology. These investigations could ultimately lead to 



100 

development of therapeutic agents that target disease processes involving chemokines and 
chemokine receptors. 






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BIOGRAPHICAL SKETCH 

Yan Jiang was born in May 1964 in Shanghai. People's Republican of China. 
After finished her 1 1 years of precollege education, she went to medical school of 
Shanghai Medical University in 1981 and got her medical degree six years later. In 1987, 
she started her first post-graduate education as a resident in Department of radiation 
oncology in Shanghai Cancer Hospital. During 5 years of her residency, she learned 
everything needed to be a good doctor and enjoyed the experience. However, close to the 
end of her residency, she realized that there was not enough challenge in the future if she 
stayed in the same field as a radiation oncologist; it was time to try something new. She 
decided to come to the States with her husband and start a new career as a medical 
research scientist. The begining was painful as she put herself in a foreign country with 
very little background in research and spoke very little English. But she managed to 
survive as time went by. In 1994, she was accepted and went to graduate school in 
department of Pharmacology and Therapeutics of the College of Medicine, University of 
Florida, pursuing a Ph.D. degree. She has had a fun, frustrating but rewarding four and 
half years during which she became a "Gatorized" Chinese. Now, it's time for her to 
move on. 



126 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




iy K. Harrison, Chair 
kssjistant Professor of Pharmacology and 
Therapeutics 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy 




Wolfgang J. Streit 

Associate Professor of Neuroscience 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




lomas C. Rowe 
Associate Professor of Pharmacology and 
Therapeutics 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Joel Schjffenbauer 
Associate Professor of Molecular 
Genetics and Microbiology 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Jo,Hn M. Petitt6 

Associate Professor of Neuroscience 



This dissertation was submitted to the Graduate Faculty of the College of 
Medicine and to the Graduate School and was accepted as partial fulfillment of the 
requirements for the degree of Doctor of Philosophy. 



December 1998 



dtJ^uBL- 



Dean, College of Medicine 






















UNIVERSITY OF FLORIDA 



3 1262 08555 3112